14-3-3 zeta over-expression as a poor prognosis factor, and a therapeutic target in multiple cancer types

ABSTRACT

Methods of determining prognosis in a subject with a hyperproliferative disease, including determining expression and/or function of 14-3-3 zeta in the subject, are disclosed. Also disclosed are methods of making a pharmaceutical agent that modulates apoptosis, including the steps of obtaining one or more candidate, testing the one or more candidate substances to determine their ability to modulate the expression and/or function of 14-3-3 zeta, selecting a candidate substance determined to modulate the expression and/or function of 14-3-3 zeta, and making a pharmaceutical composition that includes the selected candidate substance. In addition, methods of treating a subject with a hyperproliferative disease, including making a pharmaceutical agent by the methods set forth herein, and administering the pharmaceutical agent to a subject, are disclosed. The hyperproliferative disease can be cancer, such as breast cancer.

This application claims the benefit of U.S. Provisional Application No.60/501,786, filed on Sep. 10, 2003, which is incorporated by referencein its entirety.

The government owns rights in the present invention pursuant to grantnumber USAMRMC DAMD-17-01-0306 from the U.S. Army.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cancer biologyand molecular biology. More particularly, it concerns methods ofdetermining prognosis in a subject with a hyperproliferative diseasethat involve determining expression and/or function of 14-3-3 zeta inthe subject. It also concerns methods of making a pharmaceutical agentthat modulates apoptosis, including the steps of obtaining one or morecandidate, testing the one or more candidate substances to determinetheir ability to modulate the expression and/or function of 14-3-3 zeta,selecting a candidate substance determined to modulate the expressionand/or function of 14-3-3 zeta, and making a pharmaceutical compositionthat includes the selected candidate substance. In addition, the presentinvention concerns methods of treating a subject with ahyperproliferative disease, including making a pharmaceutical agent byany of the methods set forth herein, and administering thepharmaceutical agent to a subject.

2. Description of Related Art

The 14-3-3 proteins constitute a family of highly conserved dimericproteins that are ubiquitously expressed in eukaryotic organisms (Aitkenet al., 1992). These proteins were originally isolated in 1967 (Mooreand Perez, 1967). The name “14-3-3” is derived from the particularmigration pattern on two-dimensional DEAE-cellulose chromatography andstarch gel electrophoresis (Moore and Perez, 1967). In humans, nineisoforms have been found to be encoded by seven 14-3-3 genes. Theencoded proteins have a molecular weight of 29 kDa -31 kDa.

High levels of 14-3-3 proteins were originally shown to exist inneuronal tissue, and it was originally thought that they wereneuron-specific (Moore and Perez, 1967). However, they have now beenshown to be widely distributed and present at low levels in mostmammalian tissues. Proteins that show a high degree of similarity havebeen cloned and sequenced from a wide range of other eukaryoticorganisms including plants, insects, amphibians and yeast (Aitken etal., 1992).

The 14-3-3 family is highly conserved over a wide range of mammalianspecies, and the 14-3-3 isoforms can be found in an extremely broadrange of tissues. There are very high levels of many isoforms in braintissue, particular Purkinje cells in the cerebellum (Watanabe et al.,1991). High levels of beta and gamma isoforms are also found, the latterof which is believed to be specific for the brain (Isobe et al., 1991).There are also high levels of some isoforms in adrenal medulla andintestine, platelets, and testis (Ichimura et al., 1991). Other isoformsare expressed in spleen, skin, ear, and tongue (Aitken et al., 1992).Homologues of 14-3-3 proteins have been found in a broad range ofeukaryotic organisms and are probably ubiquitous (reviewed in Wang andShakes, 1996; Rosenquist et al., 2000).

Crystal structures of both the tau and zeta isoforms of 14-3-3 show thatthey are highly helical, dimeric proteins (Xiao et al., 1995; Liu etal., 1995). Each monomer is composed of nine anti-parallel α-helices,organized into an N-terminal and a C-terminal domain. The dimer createsa large negatively charged channel. Those regions of the 14-3-3 proteinswhich are invariant throughout all of the isoforms are mainly foundlining the interior of this channel, while the variable residues arelocated on the surface of the protein (Aitken et al., 2002). Thischannel might recognize common features of target proteins, so thespecificity of interaction of 14-3-3 isoforms with diverse targetproteins may involve the outer surface of the protein (Aitken et al.,2002). The N-terminal residues of all 14-3-3 homologues are variable,and are involved in dimer formation (Aitken et al., 2002).

The known functions of the 14-3-3 class of proteins include a wide rangeof cell signaling processes as well as development and growthregulation. The first function of this family of proteins that wasdescribed was activation of tyrosine and tryptophan hydroxylases, therate-limiting enzymes involved in catecholamine and serotoninbiosynthesis, essential for the synthesis of dopamine and otherneurotransmitters (Ichimura et al., 1988). Subsequently, it was shownthat 14-3-3 could regulate (inhibit) activity of protein kinase C (PKC)(Aitken et al., 1990; Toker et al., 1990). 14-3-3 was then found to be anovel type of chaperone protein that modulates the interaction betweencomponents of signal-transduction pathways (Aitken, 1996). Previousstudies indicated that different 14-3-3 isoforms have overlapping roleswithin cells as they bind many of the same target proteins (Yaffe,2002).

In the mid-1990's, numerous reports showed that 14-3-3 proteins couldinteract with a wide range of protein kinases, phosphatases, and othersignaling proteins (reviewed in Aitken et al., 2002), which implies that14-3-3 proteins mediate the formation of protein complexes involved insignal transduction, trafficking and secretion, perhaps to bind todifferent signaling proteins on each subunit of the dimer, as a noveltype of ‘adapter protein.’

In addition, 14-3-3 proteins play a role in suppression of apoptosis.They bind many proteins involved in regulation of apoptosis, such as BAD(Zha et al., 1996), A20 (De Valck et al., 1997), Forkhead (Brunet etal., 1999), and ASK1 (Zhang et al., 1999). Compromising 14-3-3 functionby overexpression of a competitive 14-3-3 binding peptide rendered cellsmore sensitive to apoptotic stimuli (Masters and Fu, 2001).

One of the kinases that has been shown by the inventors to interact with14-3-3 proteins is phosphatidylinositol 3-kinase (PI-3-kinase). The PI-3kinases represent a ubiquitous family of heterodimeric lipid kinasesthat are found in association with the cytoplasmic domain of hormone andgrowth factor receptors and oncogene products. PI3Ks act as downstreameffectors of these receptors, are recruited upon receptor stimulationand mediate the activation of second messenger signaling pathwaysthrough the production of phosphorylated derivatives of inositol (Fry etal., 1994). PI3Ks have also been implicated in many cellular activitiesincluding growth factor mediated cell transformation, mitogenesis,protein trafficking, cell survival and proliferation, DNA synthesis,apoptosis, neurite outgrowth and insulin-stimulated glucose transportreviewed in (Fry et al.,1994).

The PI3-kinase enzyme heterodimers most commonly consist of a 110 kD (p110) catalytic subunit associated with an 85 kD (p85) regulatorysubunit. Recently however, three smaller regulatory subunits have beenidentified, two 55 kD subunits (p55.alpha. and p55.gamma.) and one 50 kDsubunit (p50.alpha.) (Shin et al., 1998). Modulation of PI3-kinaseactivity by 14-3-3 zeta in cancer cells is not established from previousstudies. (Munday et al., 2000; Guthridge et al., 2000; Liu et al.,1996). These previous studies concerned hematopoietic rather than cancercells.

Cancer is a major cause of morbidity and mortality in the si U.S. Breastcancer, for instance, now affects as many as one in eight women duringtheir lifetime (Ries et al., 1999; Sondik 1994). In many regions of theworld, breast cancer is the more frequently occurring malignant diseasein women (Forbes, 1997). Methods of diagnosing and treating breastcancer are a major research focus in the U.S.

Although many biomarkers for breast cancers (e.g., estrogen receptor,ErbB2, Bc1-2) have been discovered during the last decades, idealprognostic factors for breast cancers are still lacking (Rogers et al.,2002). For instance, only about 30% of breast cancers overexpressc-erb2, epidermal growth factor receptor, cyclin D1, or c-myc (reviewedin Welch and Wei, 1998).

There is no clear role of 14-3-3 isoforms in human cancer. Inparticular, there has been no previous report on tumor promotingfunction of 14-3-3 isoforms in human cancers. Recently, however, loss of14-3-3 sigma gene expression was found to be a frequent event in breastcancer (Ferguson et al., 2000). The 14-3-3 sigma isoform is believed tobe responsible for instituting the G₂ cell cycle checkpoint response toDNA damage in human cells (Hermeking et al., 1997; Chan et al., 2000).Although 14-3-3 sigma has been associated with tumor suppression(Ferguson et al., 2000), this function has not been ascribed to other14-3-3 isoforms. Another study found that levels of the alpha, beta,delta, and zeta isoforms of 14-3-3 were the same in both normal andtransformed cells (Vercoutter-Edouart et al., 2001). These resultsindicate that the precise role of the 14-3-3 family of proteins in humancancer remains ill-defined.

Therefore, the identification of the precise role of 14-3-3 proteins inhuman cancer may provide valuable insight that can be applied in theclinical care of cancer patients. Detailed elucidation of anyrelationship between 14-3-3 expression and cancer could provide newforms of cancer therapy. Knowledge of a correlation between 14-3-3protein levels and cancer severity or cancer type could be applied informulating accurate prognosis of cancer patients, and could also beapplied in designing targeted therapeutic and preventive strategies.

SUMMARY OF THE INVENTION

The inventors have discovered that 14-3-3 zeta expression is elevated inmany types of human cancers, including breast cancer, sarcomas, lungcancer, liver cancer, uterine cancer, and stomach cancer. It has furtherbeen determined that 14-3-3 zeta is overexpressed in over 70% of humanbreast cancers, soft tissue sarcomas, and in many breast cancer celllines, lung cancer cell lines, and soft tissue sarcoma cell lines. Theinventors found that patients with 14-3-3 zeta-overexpressing breastcancers had significantly lower disease-free and overall survival rates.These findings demonstrate that overexpression of 14-3-3 zeta is afrequent event in multiple types of cancers, is associated with poorprognosis, and can be used as a novel molecular marker to assess theagressiveness of a variety of cancer types and the need for morevigorous therapies. In addition, 14-3-3 zeta is a novel clinicaltherapeutic target in multiple cancer types, and blocking 14-3-3 zetaexpression and/or function is an effective strategy to inhibit multipletypes of cancers.

Certain embodiments of the present invention generally pertain tomethods of determining prognosis in a subject with a hyperproliferativedisease. Determining prognosis involves a prediction of the probablecourse and outcome of a disease, or the likelihood of recovery from adisease. These methods of determining prognosis involve determiningexpression and/or function of 14-3-3 zeta in a subject. Any method ofdetermining expression and/or function of 14-3-3 zeta in a subject thatis known to those of ordinary skill in the art is encompassed within theembodiments of the invention.

In some embodiments, expression and/or function of 14-3-3 zeta ismeasured in a body fluid sample or tissue sample from the subject. Anybody fluid sample from a subject is contemplated by the embodiments ofthe present invention. For example, in certain embodiments, the bodyfluid sample is a serum sample, a plasma sample, a blood sample, acerebrospinal fluid sample, a urine sample, or a sample of fluidaspirated from a hyperproliferative lesion such as a breast lesion.Similarly, any tissue sample from a subject is contemplated by theembodiments of the present invention. In certain embodiments, forexample, the tissue sample may be tissue from a hyperproliferativelesion in a subject. The hyperproliferative lesion may be a cancer, suchas breast cancer, lung cancer, ovarian cancer, brain cancer, livercancer, cervical cancer, colon cancer, renal cancer, skin cancer, head &neck cancer, bone cancer, esophageal cancer, bladder cancer, uterinecancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicularcancer, lymphoma, or leukemia.

In some specific embodiments of the present invention, expression and/orfunction of 14-3-3 zeta is measured in a cell in a subject. Any cell ina subject is contemplated by the present invention. For example, thecell may be a cell from a hyperproliferative lesion, such as a cancer ofany of the types previously set forth. In certain particularembodiments, the cell is a breast cancer cell.

Any subject is contemplated for inclusion in the embodiments of thepresent invention. In certain specific embodiments, for example, thesubject is a human. The human may be afflicted by a hyperproliferativedisease, such as a cancer. For example, as set forth above, the cancermay be breast cancer, lung cancer, ovarian cancer, brain cancer, livercancer, cervical cancer, colon cancer, renal cancer, skin cancer, head &neck cancer, bone cancer, esophageal cancer, bladder cancer, uterinecancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicularcancer, lymphoma, or leukemia. In some embodiments, the patient is abreast cancer patient.

Any method of determining expression and/or function of 14-3-3 zetaknown to those of ordinary skill in the art is contemplated forinclusion in the embodiments of the present invention. For example,determining expression and/or function of 14-3-3 zeta may be measured bywestern blot analysis, immunohistochemistry, and/or protein array. Insome embodiments, expression and/or function of 14-3-3 is measured bydetermining mRNA transcription as an indirect measure of 14-3-3 zetaprotein expression in a cell. In other embodiments, measuring 14-3-3zeta expression is determined by measuring gene copy number of 14-3-3zeta as an indirect measure of 14-3-3 zeta protein expression in a cell.Gene copy number may be measured by any method known to those ofordinary skill in the art, such as FISH, array CGH, Southern blotanalysis, and/or quantitative real time PCR.

Certain other embodiments of the present invention are generallyconcerned with methods of making a pharmaceutical agent that modulatesapoptosis, including the steps of: (1) obtaining one or more candidatesubstances; (2) testing the one or more candidate substances todetermine their ability to modulate the expression and/or function of14-3-3 zeta; (3) selecting a candidate substance determined to modulatethe expression and/or function of 14-3-3 zeta; and (4) making apharmaceutical composition comprising the selected candidate substance.Any candidate substance is contemplated by the present invention. Forexample, the candidate substance may be a small molecule, a peptide, apolypeptide, a protein, a polynucleotide, antibody, or an si RNA. Insome embodiments, the candidate substance is si RNA.

In some embodiments, the methods of the present invention furtherinclude administering the pharmaceutical agent to a subject having ahyperproliferative disease. The hyperproliferative disease may be acancer, such as a cancer of any of the types set forth above. In someembodiments, the cancer is breast cancer.

In some embodiments, the present invention includes contacting thecandidate substance with a cell and measuring expression and/or functionof 14-3-3 zeta in the cell. Any cell is contemplated for inclusion inthe present invention. For example, the cell may be a cell in a subject,such as a human. The human may be a patient with cancer, such as apatient with breast cancer.

Any method of testing the one or more candidate substances to determinetheir ability to modulate the expression and/or function of 14-3-3 zetaknown to those of ordinary skill in the art is contemplated forinclusion in the present invention. For example, testing the one or morecandidate substances to determine their ability to modulate theexpression and/or function of 14-3-3 zeta may further be defined astesting the one or more candidate substances to determine their abilityto modulate the interaction of 14-3-3 zeta with PI-3-kinase. Ability tomodulate any mechanism of interaction of 14-3-3 zeta with PI-3-kinase iscontemplated by the methods of the present invention. For example,testing of the interaction of 14-3-3 zeta with the p85 subunit ofPI3-kinase is contemplated, as is testing of the interaction of 14-3-3zeta with serine 83 of the p85 subunit of PI-3-kinase.

Some embodiments of the present invention pertain to methods of treatinga subject with a hyperproliferative disease that involve making apharmaceutical agent by any of the methods set forth above, andadministering the pharmaceutical agent to a subject. Although anysubject is contemplated for inclusion in the methods of the presentinvention, in some embodiments the subject is a patient with cancer. Thesubject may have any type of cancer. Some examples of cancer types havebeen set forth above. For example, in certain embodiments, the cancer isbreast cancer.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claim(s), whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D. Specific silencing of 14-3-3 zetareduces cell proliferation by inducing a G₁ arrest. FIG. 1A: Colonyformation in soft agar of 435.neo (▪) and 435.1433 zeta (□) cells inindicated serum concentrations. Colonies were counted and the foldincrease in colony numbers determined. The data show that increased14-3-3 zeta expression in 435.1433 zeta cells leads to increased colonyformation. FIG. 1B: 435.neo (▪) and 435.1433 zeta (□) cells were exposedto γ-radiation, and apoptosis was assayed by FACS analyses ofannexin-positive cells at 0 hour and after 72 hours. The data show thathigher 14-3-3 zeta expression in 435.1433 zeta cells confers resistanceto IR-induced apoptosis. FIG. 1C: MDA-MB-435 cells were transfected bymock (⋄), control (□), or 14-3-3 zeta (▪) siRNA. Cells were plated in24-well plates in either 10% or 0.5% serum and counted on the indicateddays to determine the growth rate. FIG. 1D: The indicated cell lineswere treated with control (□) or 14-3-3 zeta (▪) siRNAs. The cells werecollected 72 hours post-transfection and cell cycle G₁ percentage weredetermined by FACS analysis. These data show that downregulation of14-3-3 zeta by siRNA leads to reduced cell proliferation by inducing aG₁ arrest.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F. Silencing 14-3-3zeta by siRNA sensitizes cells to stress-induced apoptosis and reducestumorigenic properties of breast cancer cells. FIG. 2A: MCF-7 cells weretreated with 14-3-3 zeta (▪), or control (□) siRNAs. Forty-eight hourspost-transfection, the cells were split into multiple plates, allowed toattach for 24 hours, and replenished with serum-free media. At theindicated time points, apoptotic cells were identified by annexin-Vstaining and FACS analysis. FIG. 2B: MCF-7 breast cancer cells and HeLacervical cancer cells were treated as discussed in Example 1 andpropidium iodide staining and FACS analysis identified the sub-G1 phase.FIG. 2C: MCF-7 cells were treated as in FIG. 2A except a duplicate wellof cells were also treated with Z-VAD-FMK caspase inhibitor for 24 hoursin serum-free media. The annexin-positive cells were analyzed as in FIG.2A. FIG. 2D: MCF-7 and MDA-MB-435 cells were transfected with 14-3-3zeta (▪) or control (□) siRNAs. Colony formation in 10% FBS soft agarwas determined by crystal violet staining on day 14. FIG. 2E and FIG.2F: MDA-MB-435 cells were treated with mock (⋄), control (□) or 14-3-3zeta (▪) siRNA, and 1×10⁶ cells were injected into the mammary fat padsof SCID mice (10 mice/group). In FIG. 2E, tumor onset was determined bymeasurable tumors on the indicated days. In FIG. 2F, the tumor volumewas determined by measuring the length and width of the tumors on theindicated days.

FIG. 3. Overexpression of 14-3-3 zeta correlates to poor survival inbreast cancer patients. 14-3-3 zeta protein expression was measured byimmunohistochemical (IHC) analysis in primary invasive breast carcinomasfrom 107 patients, who had chemotherapies after mastectomy, with amedian follow-up time of 72 months. Representative 14-3-3 zeta IHCstaining in breast cancer specimens (n=107) was graded as negative,weakly positive, moderately positive, or strongly positive for 14-3-3zeta overexpression. Benign breast epithelial cells and stromalcomponents were negative or weakly positive for 14-3-3 zeta staining.Overall and disease-free survival curve of patients with tumors havinglow (negative, weak, moderately positive) (—) (n=59) or high (stronglypositive) (---) (n=48) 14-3-3 zeta expression.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D. 14-3-3 zeta binds to p85 andmodulates PI3K activity and Akt activation. FIG. 4A: MCF-7 and H1299cells were stably transfected with HA-14-3-3 zeta expression vector.Cells were either grown in 10% FBS/DMEM media (+), serum starved for 18hours (−), or serum starved for 18 hours then stimulated with 10%FBS/DMEM media for 10 or 30 minutes (10′, 30′), lysed andimmunoprecipitated with HA antibody to bring down 14-3-3 zeta and 14-3-3zeta binding proteins. Interaction of HA-14-3-3 zeta and endogenous p85was analyzed by western blotting using anti-p85. Immunoprecipitationwith IgG was included as a negative control. FIG. 4B: MCF-7 cells stablytransfected with HA-14-3-3 zeta were treated as in FIG. 4A. −/+indicates cells were serum starved for 18 hr and then stimulated for 10minutes. Cell lysates were immunoprecipitated with antibodies to p85,p110 or IgG as control. Interactions of endogenous p85 and p110 withHA-14-3-3 zeta were analyzed by western blotting using anti-HA to detectbound 14-3-3 zeta. These data indicates 14-3-3 zeta can associate withboth subunits of PI3K. FIG. 4C: MCF-7 cells were treated with siRNA to14-3-3 zeta or a control. Cells were serum starved for 18 hours thenstimulated with Heregulin for 10 minutes (−/+ HRG), lysed andimmunoprecipitated with a general phospho-tyrosine antibody (PY20) topull down activated receptor tyrosine kinases and associated proteins.Immunoprecipitates were subjected to PI-3K assays. Kinase activity wasdetermined by production of PIP3. The data show downregulation of 14-3-3zeta reduces PI3K activity when stimulated with Heregulin. FIG. 4D:MCF-7 and MDA-MB-435 cells were treated with siRNA to 14-3-3 zeta or acontrol. Forty-eight hours post-transfection, the cells were split intomultiple plates, allowed to attach for 24 hours and replenished withserum-free media for 18 hours. Cells were re-stimulated with 10%FBS/DMEM media and collected at the indicated time points (minutes). Aktactivity was determined by measuring Akt phosphorylation status usingwestern blot analysis with a phospho-serine 473 Akt antibody. Immunoblotof Akt was used as a loading control. The data indicate 14-3-3 zeta isimportant for Akt activation by serum.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E. Mutation of serine 83 onp85 reduces 14-3-3 zeta association, reduces PI3K activity andsensitizes cells to apoptosis under serum-free conditions. FIG. 5A:MCF-7 cells were stably transfected with HA-14-3-3 zeta and histidinetagged (HIS) wild type p85 (p85^(wt)) or p85 with serine 83 mutated toalanine (p85^(S83A)) or an empty vector. Expression levels weredetermined by western blot analysis using p85 antibody (upper panel) andHA antibody (lower panel). FIG. 5B: Stably transfected MCF-7 cells inFIG. 5A were serum starved for 18 hours and stimulated with 10% FBS/DMEMmedia, lysed, and immunoprecipitated with p85 antibody or HIS antibodyto bring down total p85 or HIS-tagged p85. Association of HA-14-3-3 zetawith the indicated p85 proteins was determined by western blot analysisusing HA antibody (lower panel) and IP efficiency was determined bywestern blot with p85 antibody. The data indicate that serine 83 isimportant for 14-3-3 zeta association with p85. FIG. 5C: MCF-7 cellsstably transfected as in FIG. 5A were grown in 10% FBS/DMEM media, lysedand immunoprecipitated with a phospho-tyrosine antibody (PY20) to pulldown activated receptor tyrosine kinases and associated proteins.Immunoprecipitates were subjected to PI3K assays (left panel). PI3Kactivity was determined by production of PIP3. The graph representsrelative percent PI3K activity for the indicated p85 proteins (rightpanel). Data shown is a quantitative analysis of PI-3K activitydetermined by dividing the intensity of the PIP3 signal by the intensityof the PIP3 and origin signals. FIG. 5D: MCF-7 cells in FIG. 5A weremaintained in 10% FBS/DMEM media. Akt activity was measured by westernblot analysis using phospho-serine 473 Akt antibody. Immunoblot of Aktwas used as loading control. Relative intensity of phospho-Akt wascalculated by dividing the band intensity of phospho-Akt by Akt. Thevector phospho-Akt/Akt ratio was set as in FIG. 4. FIG. 5E: MCF-7 cellsin FIG. 5A were serum starved for the indicated time periods. Apoptoticcells were identified by TUNEL and FACS analysis. The data indicate that14-3-3 zeta association with p85 is necessary for complete activation ofPI-3K and Akt and disruption of 14-3-3 zeta association with p85sensitizes cells to apoptosis induced by serum starvation.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention seeks to exploit the inventors' discovery byproviding for methods of determining prognosis in a subject with ahyperproliferative disease, such as cancer, that involve determining14-3-3 zeta expression and/or function in a subject. For example,increased 14-3-3 zeta expression in a biopsy sample from a tumor of asubject with cancer may be used to indicate poorer prognosis, and theneed for a more aggressive therapeutic regimen.

The present invention also provides for methods of making apharmaceutical agent that modulates apoptosis, involving the steps of:(1) obtaining one or more candidate substances; (2) testing the one ormore candidate substances to determine their ability to modulate theexpression and/or function of 14-3-3 zeta; (3) selecting a candidatesubstance determined to modulate the expression and/or function of14-3-3 zeta; and (4) making a pharmaceutical composition that includesthe selected candidate substance. The present invention also providesfor methods of treating a subject with a hyperproliferative disease,including making a pharmaceutical agent by any of the methods set forthherein, and administering the pharmaceutical agent to the subject.

A. 14-3-3 Zeta Protein

Throughout this application, the term “14-3-3 zeta” is intended to referto the exemplified 14-3-3 zeta molecules as well as all 14-3-3 zetahomologues from other species. The full-length amino acid sequence ofthe human 14-3-3 zeta protein is provided herein, and is designated SEQID NO:1 (GenBank Accession Number NP_(—)003397).

The term “14-3-3 zeta” refers to both “wild-type” and “mutant” 14-3-3zeta. Wild-type 14-3-3 zeta refers to a 14-3-3 zeta molecule havingnormal 14-3-3 zeta activity, as exemplified by SEQ ID NO:1. Mutant14-3-3 zeta includes sequence variants of 14-3-3 zeta that may or maynot have reduced 14-3-3 zeta activity.

Inherent in the definition of sequence variants is the concept thatthere is a limit to the number of amino acid changes that may be madewithin a defined portion of the molecule and still result in a moleculewith an acceptable level of equivalent biological activity, i.e.,ability of 14-3-3 zeta to modulate cell death. Sequence variants arethus defined herein as those polypeptides in which certain, not most orall, of the amino acids may be substituted. Of course, a plurality ofdistinct proteins/polypeptides/peptides with different substitutions mayeasily be made and used in accordance with the invention.

Amino acid sequence variants of the polypeptide can be substitutionalmutants or insertional mutants. Insertional mutants typically involvethe addition of material at a non-terminal point in the peptide. Thismay include the insertion of a few residues; an immunoreactive epitope;or simply a single residue. The added material may be modified, such asby methylation, acetylation, and the like. Alternatively, additionalresidues may be added to the N-terminal or C-terminal ends of thepeptide.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, or example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

Throughout this application, the terms “14-3-3 zeta activity,” “14-3-3zeta expression,” and “14-3-3 zeta function” refer to the activity,expression, and function of the 14-3-3 zeta protein. As used herein inthis application, 14-3-3 zeta protein includes 14-3-3 zeta proteins fromany and all species, including the human protein. 14-3-3 zeta activityor 14-3-3 zeta function refers to the functional activity of the 14-3-3zeta protein. Any method known to those of skill in the art can be usedto quantitate functional activity of the 14-3-3 protein. For example,increased cell death can be used as a measure of decreased 14-3-3 zetaactivity.

14-3-3 zeta expression refers to expression of the 14-3-3 protein.14-3-3 zeta expression can be measured directly, such as by proteinassays. For example, 14-3-3 zeta expression can be measured by westernblot analysis, immunohistochemistry, protein array, or any method knownto those of skill in the art. Alternatively, 14-3-3 expression can bemeasured indirectly, such as by measurement of 14-3-3 zeta mRNAtranscription and/or stability, measurement of gene copy number of the14-3-3 zeta gene in a cell, or any method known to those of skill in theart.

In certain embodiments, the 14-3-3 zeta protein may be purified.Generally, “purified” will refer to a 14-3-3 zeta protein compositionthat has been subjected to fractionation to remove various otherproteins, polypeptides, or peptides, and which composition substantiallyretains its activity. Purification may be substantial, in which the14-3-3 zeta is the predominant species, or to homogeneity, whichpurification level would permit accurate degradative sequencing.

The 14-3-3 zeta protein purified from a natural source or fromrecombinantly-produced material. Those of ordinary skill in the artwould know how to produce these polypeptides from recombinantly-producedmaterial. This material may use the 20 common amino acids in naturallysynthesized proteins, or one or more modified or unusual amino acids.

Any method known to those of ordinary skill in the art can be used topurify the 14-3-3 zeta protein. Purification methods, for example, mayresult in 14-3-3 zeta protein that has been purified to at least about20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% and up to about 100% homogeneity. Alternatively, 14-3-3 zetaprotein may be purified to at least or at most about 20%-95%, 30%-90%,40%-80%, 50%-75%, 20%-50%, 50%-70%, 50%-90%, 70%-90% and ranges inbetween. The term “homogeneity” refers to the percent of 14-3-3 zetaprotein as compared to the total amount of protein (by molecule). Theterm “about” refers to the imprecision of determining protein amounts,and is intended to include at least one standard deviation of error forany particular assay or calibration for measuring protein concentration.For example, if protein concentration and homogeneity is measured by gelelectrophoresis with coomassie gel staining, 14-3-3 zeta protein thathas been purified to at least about 25% homogeneity means that thesample placed on the gel is at least 25% 14-3-3 zeta, as compared tototal protein concentration by molecule, plus or minus the standarddeviation for a protein gel stained with coomassie dye.

B. Candidate Substances

The present invention includes methods of making a pharmaceutical agentthat modulates apoptosis, including the steps of obtaining one or morecandidate, testing the one or more candidate substances to determinetheir ability to modulate the expression and/or function of 14-3-3 zeta,selecting a candidate substance determined to modulate the expressionand/or function of 14-3-3 zeta, and making a pharmaceutical compositionthat includes the selected candidate substance.

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). Apoptosis refers to, forexample, cell shrinkage, chromatin condensation, nucleus concentration,disappearance of microvillus on the cell surface, plasma membraneblebbing, apoptotic body formation, gap between peripheral cellsaccompanied with cell shrinkage, and removal by phagocytes (JapanClinic, vol.54, No. 7 (1996)). Apoptosis or programmed cell death playsan important role in individual development and homeostasis maintenancein a living body. It has been gradually made clear that abnormality ofapoptosis causes diseases such as cancers, autoimmune diseases andnervous diseases.

Any method of testing a candidate substance known to those of ordinaryskill in the art is contemplated by the present invention. For example,assays to assess ability of the candidate substance to modulateexpression and/or function of 14-3-3 zeta are contemplated. The assaysmay comprise random high-throughput screening of large libraries ofcandidate substances. Alternatively, the assays may be used to focus onparticular classes of compounds selected with an eye towards structuralattributes that are believed to make them more likely to modulate thefunction of this molecule. The assays may be cell-free assays, in vitroassays, in cyto assays, in vivo assays, or any assay technique known tothose of skill in the art.

By function, it is meant that one may assay for effects on function of14-3-3 zeta. For example, a candidate substance can be analyzed for itsability to bind to 14-3-3 zeta. Alternatively, the candidate substancecan be analyzed for its ability to interfere with the ability of 14-3-3zeta to prevent cell death. For example, the cell can be analyzed forapoptosis and other histological changes associated with cell death.

A modulator of the expression and/or function of 14-3-3 zeta is anysubstance that can inhibit or promote the expression and/or function of14-3-3 zeta.

The candidate substance can be a candidate substance suspected of eitherinhibiting or promoting apoptosis.

1. Modulators

As used herein the term “candidate substance” refers to any moleculethat may potentially inhibit or enhance apoptotic cell death. Thecandidate substance may be a protein or fragment thereof, a smallmolecule, an antibody, or even a polynucleotide.

The candidate substance may also be small interfering RNA (“siRNA”)molecules. siRNA's for mammalian systems are typically composed ofdouble-stranded RNA with 19 to 28, preferable 19 to 23, nucleotide RNAstrands, a two nucleotide overhand at the 3′ end and an optional 5′phosphate group (Yang et al., 2001; Elbashir et al., 2002). Such siRNA'sprovide a highly active and selective method for reducing the expressionof targeted genes by utilizing the RNA interference post-translationalgene silencing pathway. Interference of gene expression by interferingRNA is recognized as a naturally occurring mechanism for silencingalleles during development in plants, invertebrates and vertebrates.

It may prove to be the case that the most useful pharmacologicalcompounds will be compounds that are structurally related to 14-3-3 zetaprotein, i.e., mimics. Using lead compounds to help develop improvedcompounds is known as “rational drug design” and includes not onlycomparisons with known inhibitors and activators, but predictionsrelating to the structure of target molecules.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or target compounds. By creating suchanalogs, it is possible to fashion drugs, which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for a target molecule, or a fragment thereof. This could beaccomplished by x-ray crystallography, computer modeling or by acombination of both approaches.

It also is possible to use antibodies to ascertain the structure of atarget compound activator or inhibitor. In principle, this approachyields a pharmacore upon which subsequent drug design can be based. Itis possible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

Candidate compounds may include compounds isolated from natural sources,such as animals, bacteria, fungi, plant sources, including leaves andbark, and marine samples may be assayed as candidates for the presenceof potentially useful pharmaceutical agents. It will be understood thatthe pharmaceutical agents to be screened could also be derived orsynthesized from chemical compositions or man-made compounds. Thus, itis understood that the candidate substance identified by the presentinvention may be peptide, polypeptide, polynucleotide, small moleculeinhibitors or any other compounds that may be designed through rationaldrug design starting from known inhibitors or stimulators.

Other suitable modulators include antisense molecules, ribozymes, andantibodies (including single-chain antibodies or expression constructscoding thereof), each of which would be specific for a given targetmolecule. Such compounds are described in greater detail elsewhere inthis document. For example, an antisense molecule that bound to atranslational or transcriptional start site, or splice junctions, wouldbe ideal candidate inhibitors.

In addition to the modulating compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of themodulators. Such compounds, which may include peptidomimetics of peptidemodulators, may be used in the same manner as the initial modulators.

A modulator (i.e., inhibitor or activator) according to the presentinvention may be one which exerts its inhibitory or activating effectupstream, downstream or directly on the function of the 14-3-3 zetaprotein. Regardless of the type of inhibitor or activator identified bythe present screening methods, the effect of the inhibition or activatorby such a compound results in an altered expression and/or function of14-3-3 zeta compared to the absence of the added candidate substance.

2. In vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays can be run quickly and in large numbers, thereby increasing theamount of information obtainable in a short period of time. A variety ofvessels may be used to run the assays, including test tubes, plates,dishes and other surfaces such as dipsticks or beads. One example of acell free assay is a binding assay. While not directly addressingfunction, the ability of a candidate substance to modulate 14-3-3 zetaexpression and/or function is strong evidence of a related biologicaleffect. For example, binding of a molecule to a target may, in and ofitself, be inhibitory, due to steric, allosteric or charge-chargeinteractions. The target may be either free in solution, fixed to asupport, expressed in or on the surface of a cell. Either the target orthe compound may be labeled, thereby permitting determining of binding.Usually, the target will be the labeled species, decreasing the chancethat the labeling will interfere with or enhance binding. Competitivebinding formats can be performed in which one of the agents is labeled,and one may measure the amount of free label versus bound label todetermine the effect on binding.

A technique for high throughput screening of compounds is described inWO 84/03564, U.S. Pat. No. 6,457,809, U.S. Pat. No. 6,406,921, and U.S.Pat. No. 5,994,131. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic or some other surface.Bound polypeptide is detected by various methods.

3. In cyto Assays

In certain embodiments of the present invention, the method of making apharmaceutical agent that modulates apoptosis involves contacting thecandidate substance with a cell. The cell can then be assayed forvarious parameters associated with modulation of 14-3-3 zeta activity.For instance, the cells can be directly assayed for binding inhibitionof 14-3-3 zeta expression. Immunohistochemical techniques, confocaltechniques, or other techniques to assess binding are well known tothose of skill in the art.

Various cell lines can be utilized for such screening assays, includingcells specifically engineered for this purpose. Examples of cells usedin the screening assays include cancer cells, cells infected with avirus, foam cells, macrophages, neuronal cells or dendritic cells. Thecell may be a stimulated cell, such as a cell stimulated with a growthfactor. One of skill in the art would understand that the inventiondisclosed herein contemplates a wide variety of in cyto assays formeasuring parameters that correlate with modulation of 14-3-3 zetaactivity.

Depending on the assay, culture may be required. The cell may beexamined using any of a number of different physiologic assays to assessfor modulation of 14-3-3 zeta activity. Alternatively, molecularanalysis may be performed, for example, looking at protein expression,mRNA expression (including differential display of whole cell or polyARNA) and others such as measurement of 14-3-3 gene copy number.

4. In vivo Assays

In vivo assays involve the use of various animal models, includingtransgenic animals that have been engineered to have specific defects,or carry markers that can be used to measure the ability of a candidatesubstance to reach and effect different cells within the organism. Dueto their size, ease of handling, and information on their physiology andgenetic make-up, mice are a preferred embodiment, especially fortransgenics. However, other animals are suitable as well, includingrats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs,sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbonsand baboons). Assays for modulators may be conducted using an animalmodel derived from any of these species.

In such assays, one or more candidate substances are administered to ananimal, and the ability of the candidate substance(s) to alter one ormore characteristics, as compared to a similar animal not treated withthe candidate substance(s), identifies a modulator. The characteristicsmay be any of those discussed above with regard to the functionsassociated with 14-3-3 zeta (e.g., cell survival).

The present invention provides methods of screening of a candidatesubstance that can modulate death of a cancer cell. Treatment of theseanimals with test compounds will involve the administration of thecompound, in an appropriate form, to the animal. Any animal model ofcancer known to those of skill in the art can be used in the screeningtechniques of the present invention. Administration will be by any routethat could be utilized for clinical or non-clinical purposes, includingbut not limited to oral, nasal, buccal, intratumoral, or even topical.Alternatively, administration may be by intratracheal instillation,bronchial instillation, intradermal, subcutaneous, intramuscular,intraperitoneal. inhalation or intravenous injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

C. Nucleic Acids, Vectors and Regulatory Signals

The present invention involves methods of making a pharmaceutical agentthat modulates apoptosis, that involves testing one or more candidatesubstances to determine their ability to modulate expression and/orfunction of 14-3-3 zeta. In some embodiments of the present invention,the candidate substance is included in an expression cassette thatincludes a promoter, active in a particular cell, such as a cell in asubject, operably linked to a polynucleotide encoding the modulator.

Throughout this application, the term “expression cassette” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a gene andtranslation of a mRNA into a polypeptide.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule, RNA or DNA, that has been isolated free of totalgenomic nucleic acid. Therefore, a “polynucleotide encoding a modulatorof 14-3-3 zeta” refers to a nucleic acid segment that contains asequence that encodes a modulator of 14-3-3 zeta activity, yet isisolated away from, or purified and free of, total genomic DNA andproteins.

The present invention also encompasses derivatives of 14-3-3 zeta thathave certain amino acid changes in comparison to 14-3-3 zeta protein.The mRNA sequence encoding the full-length amino acid sequence of human14-3-3 zeta is provided below as SEQ ID NO:2 (GenBank Accession NumberNM_(—)003406).

The term “gene” is used for simplicity to refer to a functional protein,polypeptide, or peptide-encoding unit. As will be understood by those inthe art, this functional term includes genomic sequences, cDNAsequences, and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides, domains, peptides, fusionproteins, and mutants. The nucleic acid molecule encoding a modulator of14-3-3 zeta may comprise a contiguous nucleic acid sequence of thefollowing lengths or at least the following lengths: 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,8000, 8500, 9000, 9500, 10000, 11000, 12000 or more nucleotides,nucleosides, or base pairs, or any number of sequences that areencompassed in between each of these specified numbers of sequences.Such sequences may be complementary to SEQ ID NO:2 (14-3-3 zeta-encodingsequence).

The candidate substances of the present invention may include isolatedDNA segments and recombinant vectors incorporating DNA sequences thatencode a modulator of 14-3-3 zeta. In certain embodiments, the DNAsegment may substantially correspond to a portion of SEQ ID NO:2.Accordingly, sequences that have about 70%, about 75%, about about 80%,85%, about 90%, or about 95%, and any range derivable therein, such as,for example, about 70% to about 80%, and more preferably about 81% andabout 90%; or even more preferably, between about 91% and about 99%; ofamino acids that are identical or functionally equivalent to the aminoacids of SEQ ID NO:1 are contemplated by the present invention.

Vectors of the present invention are designed, primarily, to transformcells with a therapeutic gene encoding a modulator of 14-3-3 zeta inwhich the gene is under the control of regulated eukaryotic promoters(i.e., inducible, repressable, tissue specific).

The term “promoter” will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. One of ordinary skill in the art would befamiliar with use of promoters. Any promoter familiar to one of ordinaryskill in the art is contemplated by the present invention.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins. One of ordinary skill in the art would be familiar with use ofenhancers, and the types of enhancers that are available.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

Another signal that may prove useful is a polyadenylation signal. Suchsignals may be obtained from the human growth hormone (hGH) gene, thebovine growth hormone (BGH) gene, or SV40. One of ordinary skill in theart would be familiar with use of polyadenylation signals.

The use of internal ribosome binding sites (IRES) elements are used tocreate multigene, or polycistronic, messages. IRES elements are able tobypass the ribosome scanning model of 5-methylatd cap-dependenttranslation and begin translation at internal sites (Pelletier andSonenberg, 1988). One of ordinary skill in the art would be familiarwith techniques involving use of IRES elements.

In any event, it will be understood that promoters are DNA elementswhich when positioned functionally upstream of a gene leads to theexpression of that gene. Most transgene constructs of the presentinvention are functionally positioned downstream of a promoter element.

D. Gene Transfer

1. Viral Vectors

Certain embodiments of the present invention pertain to methods ofmaking a pharmaceutical agent that modulates apoptosis and methods oftreating a subject using one of the pharmaceutical agents of the presentinvention. In certain embodiments, the selected candidate substance thatis identified in the claimed methods of making a pharmaceutical agent isan expression cassette carried in a viral vector that is capable ofmodulating the expression and/or function of 14-3-3 zeta. A “viralvector” is meant to include those constructs containing viral sequencessufficient to (a) support packaging of the expression cassette and (b)to ultimately express a recombinant gene construct that has been clonedtherein.

a. Adenoviral Vectors

One method for delivery of the recombinant DNA involves the use of anadenovirus expression vector. Although adenovirus vectors are known tohave a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors.

Adenoviruses are currently the most commonly used vector for genetransfer in clinical settings. Among the advantages of these viruses isthat they are efficient at gene delivery to both nondividing an dividingcells and can be produced in large quantities. In many of the clinicaltrials for cancer, local intratumor injections have been used tointroduce the vectors into sites of disease because current vectors donot have a mechanism for preferential delivery to tumor.

The vector comprises a genetically engineered form of adenovirus.Knowledge of the genetic organization or adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz,1992). In contrast to retrovirus, the adenoviral infection of host cellsdoes not result in chromosomal integration because adenoviral DNA canreplicate in an episomal manner without potential genotoxicity. Also,adenoviruses are structurally stable, and no genome rearrangement hasbeen detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP (located at 16.8 m.u.), is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1992).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

The adenovirus vector may be replication defective, or at leastconditionally defective, the nature of the adenovirus vector is notbelieved to be crucial to the successful practice of the invention. Theadenovirus may be of any of the 42 different known serotypes orsubgroups A-F. Adenovirus type 5 of subgroup C is the preferred startingmaterial in order to obtain the conditional replication-defectiveadenovirus vector for use in the present invention. This is becauseAdenovirus type 5 is a human adenovirus about which a great deal ofbiochemical and genetic information is known, and it has historicallybeen used for most constructions employing adenovirus as a vector.

Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo. This group ofviruses can be obtained in high titers, e.g., 10⁹-10¹¹ plaque-formingunits per ml, and they are highly infective. The life cycle ofadenovirus does not require integration into the host cell genome. Theforeign genes delivered by adenovirus vectors are episomal and,therefore, have low genotoxicity to host cells. No side effects havebeen reported in studies of vaccination with wild-type adenovirus (Couchet al., 1963; Top et al., 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

b. Retroviral Vectors

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990). One of ordinary skill in the art would be familiar withconstruction and use of retroviral vectors.

C. AAV Vectors

Adeno-associated virus (AAV) is an attractive vector system for use inthe present invention as it has a high frequency of integration and itcan infect nondividing cells, thus making it useful for delivery ofgenes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has abroad host range for infectivity (Tratschin, et al., 1984; Laughlin, etal., 1986; Lebkowski, et al., 1988; McLaughlin, et al., 1988), whichmeans it is applicable for use with the present invention. Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated hereinby reference. One of ordinary skill in the art would be familiar withgeneration and use of AAV vectors.

d. Other Viral Vectors

Other viral vectors are familiar to those of ordinary skill in the art,and may be employed as constructs in the present invention. Examplesinclude herpesvirus vectors and vaccinia virus vectors. Vectors derivedfrom viruses such as poxvirus may be employed. A molecularly clonedstrain of Venezuelan equine encephalitis (VEE) virus has beengenetically refined as a replication competent vaccine vector for theexpression of heterologous viral proteins (Davis et al., 1996). Studieshave demonstrated that VEE infection stimulates potent CTL responses andhas been sugested that VEE may be an extremely useful vector forimmunizations (Caley et al., 1997). It is contemplated in the presentinvention, that VEE virus may be useful in targeting dendritic cells. Inother embodiments of the present invention, lentiviral vectors or poxviral vectors are used. One of ordinary skill in the art would befamiliar with the wide range of viral vectors that are available for usein the claimed invention.

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

e. Gene Delivery Using Modified Viruses

A polynucleotide may be housed within a viral vector that has beenengineered to express a specific binding ligand. The virus particle willthus bind specifically to the cognate receptors of the target cell anddeliver the contents to the cell. A novel approach designed to allowspecific targeting of retrovirus vectors was developed based on thechemical modification of a retrovirus by the chemical addition oflactose residues to the viral envelope. This modification can permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

2. Nonviral Vectors

a. Examples of Non-Viral Vectors

Several non-viral methods for the transfer of expression vectors intocells also are contemplated by the present invention. These includecalcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen andOkayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), directmicroinjection (Harland and Weintraub, 1985), DNA-loaded liposomes(Nicolau and Sene, 1982; Fraley et al., 1979) and liofectamine-DNAcomplexe, cell sonication (Fechheimer et al., 1987), gene bombardmentusing high velocity microprojectiles (Yang et al., 1990), polycations(Boussif et al., 1995) and receptor-mediated transfection (Wu and Wu,1987; Wu and Wu, 1988). Some of these techniques may be successfullyadapted for in vivo or ex vivo use. One of ordinary skill in the artwould be familiar with the use of nonviral vectors, and techniquesinvolving non-viral vectors.

E. Methods of Measuring Protein Expression

The present invention concerns methods of measuring prognosis in asubject with a hyperproliferative disease that include obtaining a cellor a body fluid sample from the subject and measuring increasedexpression and/or function of 14-3-3 zeta in the cell. Any method knownto those of skill in the art can be used to measure increased expressionand/or function of 14-3-3 zeta in the cell. A wide variety of techniquesto measure protein expression in a cell or in a body fluid sample areknown and familiar to one of ordinary skill in the art. Examples ofthese techniques include, but are not limited to, western blot analysis,immunohistochemistry staining, ELISA, and protein detection chips (tumormarker array).

1. Western Blot Analysis

Western blot analysis is an established technique that is commonlyemployed for analyzing and identifying proteins. The proteins are firstseparated by electrophoresis in polyacrylamide gel, then transferred(“blotted”) onto a nitrocellulose membrane or treated paper, where theybind in the same pattern as they formed in the gel. The antigen isoverlaid first with antibody, then with anti-immunoglobulin or protein Alabeled with a radioisotope, fluorescent dye, or enzyme. One of ordinaryskill in the art would be familiar with this commonly used technique forquantifying protein in a sample.

2. ELISAs

Immunoassays, in their most simple and/or direct sense, are bindingassays. Certain preferred immunoassays are the various types of enzymelinked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA)known in the art. Immunohistochemical detection using tissue sections isalso particularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and/or western blotting,dot blotting, FACS analyses, and/or the like may also be used. One ofordinary skill in the art would be familiar with use of ELISAs and otherimmunohistochemical assays.

3. Immunohistochemistry

Techniques using antibodies be used in conjunction with bothfresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks,such as blocks prepared from a tumor biopsy, prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1999; Allred etal., 1990). One of ordinary skill in the art would be familiar withimmunohistochemistry as a means to study protein expression and/orfunction.

4. Protein Array Technology

Protein array technology allows high-throughput screening for geneexpression and molecular interactions. Protein array technology isdiscussed in detail in Pandey and Mann (2000) and MacBeath and Schreiber(2000), each of which is herein specifically incorporated by reference.Protein arrays appear as new and versatile tools in functional genomics,enabling the translation of gene expression patterns of normal anddiseased tissues into protein product catalog. Protein function, such asenzyme activity, antibody specificity, and other ligand-receptorinteractions and binding of nucleic acids or small molecules can beanalyzed on a whole-genome level.

a. Protein Biochip Assays

These arrays, which contain thousands of different proteins orantibodies spotted onto glass slides or immobilized in tiny wells, allowone to examine the biochemical activities and binding profiles of alarge number of proteins at once. To examine protein interactions withsuch an array, a labeled protein is incubated with each of the targetproteins immobilized on the slide, and then one determines which of themany proteins the labeled molecule binds.

The basic construction of protein chips has some similarities to DNAchips, such as the use of a glass or plastic surface dotted with anarray of molecules. These molecules can be DNA or antibodies that aredesigned to capture proteins. Defined quantities of proteins areimmobilized on each spot, while retaining some activity of the protein.With fluorescent markers or other methods of detection revealing thespots that have captured these proteins, protein microarrays are beingused as powerful tools in high-throughput proteomics and drug discovery.

Glass slides are still widely used, since they are inexpensive andcompatible with standard microarrayer and detection equipment. However,their limitations include multiple-based reactions, high evaporationrates, and possible cross-contamination. Matrix slides offer a number ofadvantages, such as reduced evaporation and no possibility ofcross-contamination, but they are expensive. Nanochips for proteomicshave the same advantages, in addition to reduced cost and the capabilityof multiple-component reactions.

The earliest and best-known protein chip is the ProteinChip by CiphergenBiosystems Inc. (Fremont, Calif.). The ProteinChip is based on thesurface-enhanced laser desorption and ionization (SELDI) process. Knownproteins are analyzed using functional assays that are on the chip. Forexample, chip surfaces can contain enzymes, receptor proteins, orantibodies that enable researchers to conduct protein-proteininteraction studies, ligand binding studies, or immunoassays. Withstate-of-the-art ion optic and laser optic technologies, the ProteinChipsystem detects proteins ranging from small peptides of less than 1000 Daup to proteins of 300 kDa and calculates the mass based ontime-of-flight (TOF).

The ProteinChip biomarker system is the first protein biochip-basedsystem that enables biomarker pattern recognition analysis to be done.This system allows researchers to address important clinical questionsby investigating the proteome from a range of crude clinical samples(i.e., laser capture microdissected cells, biopsies, tissue, urine, andserum). The system also utilizes biomarker pattern software thatautomates pattern recognition-based statistical analysis methods tocorrelate protein expression patterns from clinical samples with diseasephenotypes.

Some systems can perform biomarker discovery in days and validation oflarge sample sets within weeks. Its robotics system accessory automatessample processing, allowing hundreds of samples to be run per week andenabling a sufficient number of samples to be run, which provides highstatistical confidence in comprehensive studies for marker discovery andvalidation.

b. Microfluidic Chip-Based Immunoassays

Microfluidics is one of the most important innovations in biochiptechnology. Since microfluidic chips can be combined with massspectrometric analysis, a microfluidic device has been devised in whichan electrospray interface to a mass spectrometer is integrated with acapillary electrophoresis channel, an injector, and a protein digestionbed on a monolithic substrate (Wang et al., 2000). This chip thusprovides a convenient platform for automated sample processing inproteomics applications.

These chips can also analyze expression levels of serum proteins withdetection limits comparable to commercial enzyme-linked immunosorbentassays, with the advantage that the required volume sample is markedlylower compared with conventional technologies.

c. Tissue Microarray Technology

Tissue microarray technology provides a high-throughput approach forlinking genes and gene products with normal and disease tissues at thecellular level in a parallel fashion. Compared with classical in situtechnologies in molecular pathology that are very time-consuming, tissuemicroarrays provide increased throughput in two ways: up to 1000 tissuespecimens can be analyzed in a single experiment, either at the DNA,RNA, or protein level; and tens of thousands of replicate tissuemicroarrays can be generated from a set of tissues. This processprovides a template for analyzing many more biomarkers than has everbeen possible previously in a clinical setting, even using archival,formalin-fixed specimens.

d. Nanoscale Protein Analysis

Most current protocols including protein purification and automatedidentification schemes yield low recoveries that limit the overallprocess in terms of sensitivity and speed. Such low protein yields andproteins that can only be isolated from limited source material (e.g.,biopsies) can be subjected to nanoscale protein analysis: a nanocaptureof specific proteins and complexes, and optimization of all subsequentsample-handling steps, leading to a mass analysis of peptide fragments.This focused approach, also termed targeted proteomics, involvesexamining subsets of the proteome (e.g., those proteins that arespecifically modified, bind to a particular DNA sequence, or exist asmembers of higher-order complexes or any combination thereof). Thisapproach is used to identify genetic determinants of cancer that altercellular physiology and respond to agonists.

F. Methods of Indirectly Measuring Gene Expression

The present invention concerns methods of measuring prognosis in asubject with a hyperproliferative disease that include obtaining a cellor a body fluid sample from the subject and measuring increasedexpression and/or function of 14-3-3 zeta in the cell. Althoughexpression and/or function of 14-3-3 zeta can be measured by one of thepreviously discussed assays, 14-3-3 zeta expression and/or function canbe measured indirectly. For example, one can measure mRNA transcriptionin a cell or measurement of gene copy number in a cell.

1. Southern Blot Analysis

One of skill in the art would be familiar with the wide range oftechniques for detecting levels of specific DNA fragments in a cell. Oneof the most well-known techniques is Southern blot analysis. Southernblot analysis is a technique for transferring DNA fragments separated byagarose gel electrophoresis to a nitrocellulose filter, on whichspecific fragments can then be detected by their hybridization toprobes, which were labeled radioactively in the original technique butare now often labeled using nonradioactive methods.

2. Fluorescence In Situ Hybridization

Fluorescence in situ hybridization is a physical mapping approach thatuses fluorescein tags to detect hybridization of probes with metaphasechromosomes and with the less-condensed somatic interphase chromatin.One of ordinary skill in the art would be familiar with this commonlyused technique.

FISH methods can be used to detect microdelections that are not visibleby standard cytogenetic banding patterns. FISH technology allows for therapid determination of whether specific genes, loci, or regions arepresent or if deletions or amplifications have occurred. The sizes ofthese regions detected by FISH are usually much larger than detectableby PCR or Southern blot, yet much smaller than visualizedmicroscopically by standard cytogenetics. This ability of FISHtechnology allows for its use in the diagnosis of cancer, prenatal,postnatal, and genetic diseases.

3. Measurement of mRNA

Gene expression may be determined by measuring the production of RNA.The RNA (e.g., mRNA) may be isolated and/or detected by methods wellknown in the art. Following detection, one may compare the results seenin a given cell line or individual (e.g., a tumor sample or a cellisolated from a body fluid sample) with a statistically significantreference group of control cells. Alternatively, one may compareproduction of RNA in cell lines transformed with the same gene operablylinked to various mutants of a promoter sequence. In this way, it ispossible to identify regulatory regions within a novel promoter sequenceby their effect on the expression of an operably linked gene.

4. DNA Arrays and Gene Chip Technology

DNA arrays and gene chip technology provides a means of rapidlyscreening a large number of DNA samples for their ability to hybridizeto a variety of single stranded DNA probes immobilized on a solidsubstrate. Specifically contemplated are chip-based DNA technologiessuch as those described by Hacia et al. (1996) and Shoemaker et al.(1996). These techniques involve quantitative methods for analyzinglarge numbers of genes rapidly and accurately. The technologycapitalizes on the complementary binding properties of single strandedDNA to screen DNA samples by hybridization. Pease et al. (1994); Fodoret al. (1991). Basically, a DNA array or gene chip consists of a solidsubstrate upon which an array of single stranded DNA molecules have beenattached. For screening, the chip or array is contacted with a singlestranded DNA sample which is allowed to hybridize under stringentconditions. The chip or array is then scanned to determine which probeshave hybridized. In a particular embodiment of the instant invention, agene chip or DNA array would comprise probes specific for chromosomalchanges evidencing the development of a neoplastic or preneoplasticphenotype. In the context of this embodiment, such probes could includesynthesized oligonucleotides, cDNA, genomic DNA, yeast artificialchromosomes (YACs), bacterial artificial chromosomes (BACs), chromosomalmarkers or other constructs a person of ordinary skill would recognizeas adequate to demonstrate a genetic change.

A variety of gene chip or DNA array formats are described in the art,for example U.S. Pat. Nos. 5,861,242 and 5,578,832 which are expresslyincorporated herein by reference. One of ordinary skill in the art wouldbe familiar with the range of techniques available using DNA arrays.

5. Array CGH

The microarray-based form of comparative genomic hybridization (arrayCGH) detects and maps DNA sequence copy number variation throughout theentire genome onto a cytogenetic map supplied by metaphase chromosomes(Kallioniemi et al., 1992). The use of metaphase chromosomes as thehybridization target has previously limited the resolution of CGH to10-20 Mb, prohibited resolution of closely speaced aberrations, and onlyallowed linkage of CGH results to genomic information and resources withcytogenetic accuracy. Array-based CGH, on the other hand, provides thecapability to map copy number aberrations relative to the genomesequence, with the resolution being determined by the spacing of theclones. In array CGN, arrays of genomic VAC, P1, cosmid or cDNA clonesare used as the hybridization target in place of the metaphasechromosomes (Solinas-Toldo et al., 1997; Pinkel et al., 1998; Pollack etal., 1999; Snijders et al., 2000). Relative copy number is then measuredat these specific loci by hybridization of fluorescently labeled testand reference DNAs as in conventional CGN (Pinkel et al., 1998). Sincethe clones used on the array contain sequence tags, their positions areaccurately known relative to the genome sequence, and genes mappingwithin regions of copy number alteration can be readily identified usinggenome databases.

Recently, array CGH has been used to identify DNA copy number variationin breast cancer tumors (Albertson, 2003). Albertson (2003), whichprovides useful information pertaining to array CGH techniques, isherein specifically incorporated by reference. In addition, alterationsin DNA copy number in bladder tumors have been recently described(Veltman et al., 2003). Veltman et al., 2003, which also provides usefulinformation pertaining to array CGH technology, is herein specificallyincorporated by reference.

6. Quantitative Real Time PCR

Quantitative real-time PCR is based on the technique of polymerase chainreaction (PCR) and can determine gene duplications or deletions (Walker,2002). Furthermore, small mutations, including single base changes, canbe identified by melting curve analysis following PCR. Real-timepolymerase chain reaction allows one to monitor the progress of the PCRas it occurs in real time. Therefore, the data is collected throughoutthe PCR process compared to regular PCR, which collects data at the endof the reaction. The real-time PCR product is detected during eachamplification cycle by labeling the PCR product as it accumulates ineach cycle with a highly specific, double-stranded fluorescent DNAbinding dye. The amount of fluorescence is either directly or indirectlyassociated with the accumulation of the newly amplified DNA. The morerapid a significant increase in fluorescence is observed indicates ahigher starting copy number of the nucleic acid target. The number ofamplicons generated is directly proportional to the increase in thereporter fluorescent signal and amplicon detection is capable ofdetecting as small as a 2-fold change. Real-time PCR methods arebecoming a favorable option for cancer marker analysis (Bernard, 2002).Recently, quantitative real-time PCR has been used to identify DNA copynumber variation in breast cancer tumors (Konigshoff, 2003).

G. Pharmaceutical Preparations and Therapeutic Methods

1. Overview

The present invention generally concerns methods of making apharmaceutical agent that modulates apoptosis, that includes the stepsof obtaining one or more candidate substances, testing the one or morecandidate substances to determine their ability to modulate theexpression and/or function of 14-3-3 zeta, selecting a candidatesubstance determined to modulate the expression and/or function of14-3-3 zeta, and making a pharmaceutical composition that includes theselected candidate substance. Certain other embodiments of the presentinvention involve methods of treating a subject with ahyperproliferative disease, that involve making a pharmaceutical agentby any of the methods set forth in this application, and administeringthe pharmaceutical agent to a subject.

2. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise aneffective amount of a selected candidate substance that has beendetermined to modulate the expression and/or function of 14-3-3 zeta,dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. Aqueous compositions of selected candidate substancesthat are gene therapy vectors are also contemplated. The phrases“pharmaceutical composition” or “pharmacologically effective” or“pharmaceutically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, or a human, as appropriate.

As used herein, “pharmaceutical composition” includes the selectedcandidate substance, plus any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. For human administration, preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biologics standards.

The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds will then generally be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, intralesional, or even intraperitonealroutes. The preparation of an aqueous composition containing an activeagent of the invention disclosed herein as a component or activeingredient will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

An agent or substance of the present invention can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptablesalts, include the acid addition salts (formed with the free aminogroups of the protein) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric, mandelic, and the like. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like. In terms of using peptide therapeuticsas active ingredients, the technology of U.S. Pat. Nos. 4,608,251;4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, eachincorporated herein by reference, may be used.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small tumorarea.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infuision, (see for example, “Remington'sPharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

The active agents disclosed herein may be formulated within atherapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligramsper dose or so. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including cremes.

One may also use nasal solutions or sprays, aerosols or inhalants in thepresent invention. Nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,the aqueous nasal solutions usually are isotonic and slightly bufferedto maintain a pH of 5.5 to 6.5. In addition, antimicrobialpreservatives, similar to those used in ophthalmic preparations, andappropriate drug stabilizers, if required, may be included in theformulation. Various commercial nasal preparations are known andinclude, for example, antibiotics and antihistamines and are used forasthma prophylaxis.

Additional formulations which are suitable for other modes ofadministration include vaginal suppositories and pessaries.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 75% of theweight of the unit, or preferably between 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor.

3. Liposomes and Nanoparticles

The use of liposomes and/or nanoparticles is also contemplated for usein the pharmaceutical compositions of the present invention. Theformation and use of liposomes is generally known to those of skill inthe art, and is also described below.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

The following information may also be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

4. Dosage

Certain embodiments of the present invention pertain to methods oftreating a subject with a hyperproliferative disease that involveadministering a pharmaceutical agent of the present invention. Aneffective amount of the therapeutic or preventive agent is determinedbased on the intended goal, for example modulation of 14-3-3 activity.The quantity to be administered, both according to number of treatmentsand dose, depends on the subject to be treated, the state of the subjectand the protection desired. Precise amounts of the therapeuticcomposition also depend on the judgment of the practitioner and arepeculiar to each individual.

In certain embodiments, it may be desirable to provide a continuoussupply of the therapeutic compositions to the patient. For topicaladministrations, repeated application would be employed. For variousapproaches, delayed release formulations could be used that providelimited but constant amounts of the therapeutic agent over an extendedperiod of time. For internal application, continuous perfusion of theregion of interest may be preferred. This could be accomplished bycatheterization, post-operatively in some cases, followed by continuousadministration of the therapeutic agent. The time period for perfusionwould be selected by the clinician for the particular patient andsituation, but times could range from about 1-2 hours, to 2-6 hours, toabout 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2weeks or longer. Generally, the dose of the therapeutic composition viacontinuous perfusion will be equivalent to that given by single ormultiple injections, adjusted for the period of time over which thedoses are administered.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

5. Treatment of Artificial and Natural Body Cavities

One of the prime sources of recurrent cancer is the residual,microscopic disease that remains at the primary tumor site, as well aslocally and regionally, following tumor excision. In addition, there areanalogous situations where natural body cavities are seeded bymicroscopic tumor cells. The effective treatment of such microscopicdisease would present a significant advance in therapeutic regimens.

Thus, in certain embodiments, a cancer may be removed by surgicalexcision, creating a “cavity.” Both at the time of surgery andthereafter (periodically or continuously), the therapeutic compositionof the present invention is administered to the body cavity. The volumeof the composition should be sufficient to ensure that the entiresurface of the cavity is contacted by the expression cassette.

In one embodiment, administration simply will entail injection of thetherapeutic composition into the cavity formed by the tumor excision. Inanother embodiment, mechanical application via a sponge, swab or otherdevice may be desired. Either of these approaches can be used subsequentto the tumor removal as well as during the initial surgery. In stillanother embodiment, a catheter is inserted into the cavity prior toclosure of the surgical entry site. The cavity may then be continuouslyperfused for a desired period of time.

6. Tracers to Monitor Gene Expression Following Administration

Because destruction of microscopic tumor cells cannot be observed, it isimportant to determine whether the target site has been effectivelycontacted with the expression cassette. This may be accomplished byidentifying cells in which the expression construct is activelyproducing the desired polypeptide product. It is important, however, tobe able to distinguish between the exogenous polypeptide and thatpresent in tumor and nontumor cells in the treatment area. Tagging ofthe exogenous polypeptide with a tracer element would provide definitiveevidence for expression of that molecule and not an endogenous versionthereof. Thus, the methods and compositions of the claimed invention mayinvolve tagging of the polypeptide encoded by the expression cassettewith a tracer element.

One such tracer is provided by the FLAG biosystem. The FLAG polypeptideis an octapeptide (AspTyrLysAspAspAspAspLys) and its small size does notdisrupt the expression of the delivered gene therapy protein. Thecoexpression of FLAG and the protein of interest is traced through theuse of antibodies raised against FLAG protein.

Other immunologic marker systems, such as the 6×His system (Qiagen) alsomay be employed. For that matter, any linear epitope could be used togenerate a fusion protein with the desired polypeptide so long as (i)the immunologic integrity of the epitope is not compromised by thefusion and (ii) the functional integrity of the desired polypeptide isnot compromised by the fusion.

7. Secondary Treatment

a. General

Certain embodiments of the claimed invention provide for a methods oftreating a subject with cancer using one of the identifiedpharmaceutical agents of the present invention. In each of theseembodiments, the subject may be receiving secondary antihyperplastictherapy. Examples of secondary antihyperplastic therapy includechemotherapy, radiotherapy, immunotherapy, phototherapy, cryotherapy,toxin therapy, hormonal therapy or surgery. Thus, the claimed inventioncontemplates use of the claimed methods and compositions in conjunctionwith standard anti-cancer therapies. The patient to be treated may be aninfant, child, adolescent or adult.

A wide variety of cancer therapies, known to one of skill in the art,may be used in combination with the compositions of the claimedinvention. Some of the existing cancer therapies and chemotherapeuticagents are described below. One of skill in the art will recognize thepresence and development of other anticancer therapies which can be usedin conjugation with the compositions comprising expression cassettes andwill further recognize that the use of the secondary therapy of theclaimed invention will not be restricted to the agents described below.

In order to increase the effectiveness of a an expression constructencoding a polypeptide that modulates 14-3-3 zeta activity, it may bedesirable to combine these compositions with other agents effective inthe treatment of hyperproliferative disease. These compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or second factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent.

Alternatively, the gene therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone may contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other. Insome situations, it may be desirable to extend the time period fortreatment significantly, however, where several d (2, 3, 4, 5, 6 or 7)to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Various combinations may be employed. For example, the inhibitor of14-3-3 zeta is therapy is “A” and the secondary agent, such as radio- orchemotherapy, is “B”:

A/B/A  B/A/B  B/B/A A/A/B  A/B/B  B/A/A A/B/B/B B/A/B/BB/B/B/A   B/B/A/B   A/A/B/B   A/B/A/B  A/B/B/A  B/B/A/AB/A/B/A   B/A/A/B   A/A/A/B   B/A/A/A  A/B/A/A  A/A/B/A

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

b. Radiotherapy

Radiotherapy include radiation and waves that induce DNA damage forexample, γ-irradiation, X-rays, UV-irradiation, microwaves, electronicemissions, radioisotopes, and the like. Therapy may be achieved byirradiating the localized tumor site with the above described forms ofradiations. It is most likely that all of these factors effect a broadrange of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

In the context of the present invention radiotherapy may be used inaddition to using the tumor cell specific-peptide of the invention toachieve cell-specific cancer therapy.

c. Surgery

Surgical treatment for removal of the cancerous growth is generally astandard procedure for the treatment of tumors and cancers. Thisattempts to remove the entire cancerous growth. However, surgery isgenerally combined with chemotherapy and/or radiotherapy to ensure thedestruction of any remaining neoplastic or malignant cells. Thus, in thecontext of the present invention surgery may be used in addition tousing the tumor cell specific-peptide of the invention to achievecell-specific cancer therapy.

In the case of surgical intervention, the compositions of the presentinvention may be used preoperatively, to render an inoperable tumorsubject to resection. Alternatively, the present invention may be usedat the time of surgery, and/or thereafter, to treat residual ormetastatic disease. For example, a resected tumor bed may be injected orperfused with a formulation comprising an expression construct. Theperfusion may be continued post-resection, for example, by leaving acatheter implanted at the site of the surgery. Periodic post-surgicaltreatment also is envisioned.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, will involve multiple doses. Typical primary tumor treatmentinvolves a 6 dose application over a two-week period. The two-weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be re-evaluated.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently may be described in terms of plaque forming units (pfu)for a viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher.

d. Chemotherapeutic Agents

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastinand methotrexate or any analog or derivative variant thereof. The term“chemotherapy” as used herein is defined as use of a drug, toxin,compound, composition or biological entity which is used as treatmentfor cancer. These can be, for example, agents that directly cross-linkDNA, agents that intercalate into DNA, and agents that lead tochromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged and are shown herein, to eventuate DNA damage leading to asynergistic antineoplastic combination. Agents such as cisplatin, andother DNA alkylating agents may be used.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis, and chromosomal segregation. Examples of thesecompounds include adriamycin (also known as doxorubicin), VP-16 (alsoknown as etoposide), verapamil, podophyllotoxin, and the like. Widelyused in clinical setting for the treatment of neoplasms, these compoundsare administered through bolus injections intravenously at doses rangingfrom 25-75 mg/m² at 21 day intervals for adriamycin, to 35-100 mg/m² foretoposide intravenously or orally.

e. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with the expression cassette. The general approach forcombined therapy is discussed below. Generally, the tumor cell must bearsome marker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155.

f. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a different expression cassette than the one disclosed herein isadministered before, after, or at the same time as the expressioncassette of the claimed invention. Delivery may comprise use of a vectorencoding polypeptide of the claimed invention in conjunction with asecond vector encoding an additional gene product such as p53.Alternatively, a single vector encoding both genes may be used. Avariety of secondary gene therapy proteins are envisioned within theinvention.

g. Other Cancer Therapies

Examples of other cancer therapies include phototherapy, cryotherapy,toxin therapy, or hormonal therapy. One of skill in the art would knowthat this list is not exhaustive of the types of treatment modalitiesavailable for cancer and other hyperplastic lesions.

G. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

RNA interference of 14-3-3 zeta in mammalian cell culture. siRNAduplexes were synthesized based on a 14-3-3 zeta-specific sequence (SEQID NO:3) and a scrambled sequence (SEQ ID NO:4) by Darmacon ResearchInc., CO. One million cells were seeded onto a 10-cm culture dish 24 hrprior to transfection in DMEM/F12 containing 10% FBS. 50 μl of siRNAduplexes (20 μM stock) were added to 750 μl Opti-MEM I (Invitrogen)containing 20 μl Plus Reagent (Invitrogen), mixed and incubated for 15min at room temperature. Then 30 μl Lipofectamine (Invitrogen) was mixedand incubated with 750 μl Opti-MEM I. After incubation, two parts weremixed and incubated for another 15 min at room temperature. Thiscombination was then assorted with 5 ml Opti-MEM I and added to cellsthat were pre-washed once with 5 ml Opti-MEM I. Five hourspost-transfection, 6.5 ml of DMEM/F12 with 20% FBS were added to thecells. The transfection solution was removed 24 hours post-transfectionand replaced with DMEM/F12 containing 10% FBS.

Immunohistochemical Staining of 14-3-3 zeta. 5 μm sections fromformalin-fixed paraffin-embedded tissue block were deparaffinized andrehydrated. The slides were subjected to heat-induced epitope retrievalin 0.01 M citrate buffer (pH 6.0). After blocked for 30 min in 3%hydrogen peroxide, and then in normal horse serum for 20 min, the slideswere incubated with 14-3-3 zeta antibody (C-16, Santa Cruzbiotechnology, Santa Cruz, Calif.) at a dilution of 1:2000 for 30 min atroom temperature. Immunodetection was performed with the LSAB2 system(DAKO, Carpinteria, Calif.). 3-3′-diaminobenzidine was used for colordevelopment, and hematoxylin was used for counterstaining.

Cytochrome C Staining. Mcf-7 cells were transfected with control or14-3-3 zeta siRNA. Forty-eight hours post transfection cells were splitto microscope slides. The next day, cells were replenished with serumfree media. At 0 and 48 hours serum free, slides were fixed and stainedwith anti-cytochrome C (Pharmingen) followed by Alexa-Fluor 594(Molecular Probes) secondary antibody and nuclei were stained with DAPI.Fluorescent signals were preserved by SlowFade Antifade Kit (MolecularProbes) and samples were analyzed by immunofluorescence microscopy and2D-deconvolution (Tan et al., 2002). Cells with diffuse cytochrome Cstaining were counted as positive for cytochrome C release.

Western Blot Analysis. Sarcomas and normal tissues were homogenized inlysis buffer (PBS containing 1% Triton X-100, 0.5% sodium deoxycholate,0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, and 0.02% sodiumazide) followed by centrifugation at 12,000×g for 10 min at 4° C. toremove cell debris. Two hundred μg of protein from both tumor and normalsamples were separated by SDS-PAGE (12% gel for p16 and CDK4 and 8% gelfor RB) and transferred onto nitrocellulose membranes (Bio-RadLaboratories, Hercules, Calif.). The membranes were blocked with 5%dried milk in TBS (0.05% Tween in PBS), probed with antibodies (1:1000dilution), washed with TBS, and probed with proper secondary antibodies(against mouse IgG or rabbit IgG). Enhanced chemiluminescence wasperformed to detect the target protein on the membrane, according to themanufacturer's protocol (Amersham Corp., Arlington Heights, Ill.). Toconfirm the results in a stringent manner, membranes were stripped andreprobed with another antibody to the same proteins of interest (p16,CDK4, and RB).

Example 2 14-3-3 Zeta Expression is Elevated in Sarcomas and PrimaryBreast Cancer

A 29 kDa protein (p29) with a polyclonal anti-p16 antibody was detectedin the sarcoma and primary breast cancer tissue specimens, presumablydue to cross-reaction. Western blot analysis using anti-p16 antibody(which recognized p29) was performed in more than 50 soft tissue sarcomasamples. Interestingly, p29 expression was elevated in 60-70% of thesarcomas as well as in more than 70% primary breast tumors compared withautologous normal tissues. The p29 protein from a sarcoma specimen waspurified to homogeneity. The p29 was purified by three steps: (a)preparative SDS-PAGE followed by gel extraction, (b) hydrophobicinteraction chromatography, and (c) preparative HPLC. Purified p29migrated as a single band of 29 kDa in a SDS-PAGE gel when stained byCoommassie blue. Microsequencing of in-gel digestion fragments ofpurified p29 revealed it as a known protein, 14-3-3 zeta. Theidentification was confirmed by immunoblotting analysis of sarcoma andbreast tumors using a 14-3-3 zeta-specific antibody, which gave anexpression patterns similar to p29.

Example 3 14-3-3 Zeta is Expressed in Multiple Tumor Types

IHC analysis of 14-3-3 zeta on a Tissue Array was conducted, whichconfirmed that 14-3-3 zeta was overexpressed in breast carcinoma. Inaddition, 14-3-3 zeta overexpression was detected in cancers of lung,liver, uterus, and stomach compared to their respective normal tissues.The tissue array slide from IMGENEX (cat# IMH-343/BA2) has 20 normaltissues and 19 different types of tumors (each tumor has one care) fromhuman origin. The normal tissues include skin, breast, spleen, skeletalmuscle, lung, liver, gallbladder, pancreas, stomach, small intestine,colon, rectum, kidney cortex, kidney medulla, bladder, prostate, uterus,placenta, umbilical cord, and fetal brain. The tumors include squamouscell carcinoma of the lung, bronchioloalveolar carcinoma of the lung,adenoid cystic carcinoma of the salivary gland, hepatocellularcarcinoma, diffuse type adenocarcinoma of the stomach, intestinal typeadenocarcinoma of the stomach, stromal sarcoma of the stomach, malignantlymphoma of the colon, mucinous carcinoma of the colon, adenocarcinomaof the rectum, malignant Schwanoma of the colon, renal cell carcinoma,poorly differentiated transitional carcinoma of the bladder, squamouscell carcinoma of the cervix, endometrial adenocarcinoma of the uterus,pheochromocytoma of the adrenal gland, metastatis adenocarcinoma of theliver (from bile duct), metastatic adenocarcinoma (from colon) of theovary, and infiltrating ductal carcinoma of the breast. These resultsare further supported by immunoblot analysis showing that 14-3-3 zetaexpression was elevated in many breast cancer, lung cancer, and sarcomacell lines compared to normal breast and lung epithelial cell lines aswell as in normal smooth muscle cells.

Example 4 14-3-3 Zeta Expression Enhances the Malignant Transformationof Cancer Cells

Elevated 14-3-3 zeta expression enhances the malignant transformation ofbreast cancer cells. MDA-MB-435 breast cancer cells were transfectedwith an expression vector for hemagglutinin (HA)-tagged 14-3-3 zeta andestablished pooled stable transfectants (435.1433 zeta) expressingexogenous 14-3-3 zeta. The 435.1433 zeta cells did not exhibit enhancedcell growth under normal culture conditions (data not shown). However,in soft agar colony formation assays, the increase of 14-3-3 zeta in435.1433 zeta cells led to a moderate increase of transforming colonieswith 10% serum and a more dramatic increase of colonies without serum(FIG. 1A). This indicates that 14-3-3 zeta can enhance theanchorage-independent growth of cancer cells. Additionally, elevatedexpression of 14-3-3 zeta conferred MDA-MB-435 cells resistance toγ-radiation-induced apoptosis (FIG. 1B).

Example 5 Blocking 14-3-3 Zeta Expression Inhibits MalignantTransformation of Cancer Cells

To further investigate the role of 14-3-3 zeta in cancer progression andto determine whether blocking 14-3-3 zeta expression would inhibitmalignant transformation and cancer development, small interfering RNA(siRNA) duplexes were used to specifically silence the expression of14-3-3 zeta in multiple cancer cell lines. After 24 hours oftransfection, the 14-3-3 zeta siRNA efficiently inhibited 14-3-3 zetaexpression without affecting the expression of 14-3-3 beta, the 14-3-3isoform most homologous to 14-3-3 zeta. This inhibition effect of 14-3-3zeta siRNA could persist for approximately 6-7 days.

14-3-3 zeta siRNA-treated cells displayed dramatic morphology changeswith cellular shrinkage and membrane blebbing, phenotypes characteristicof apoptotic cells, while the control siRNA-treated cells showed nomorphology changes under the same experimental conditions. Cell growthrate assays revealed approximately 50% growth inhibitions in MDA-MB-435and MCF-7 cells treated with 14-3-3 zeta siRNA compared to those treatedwith the control siRNA in both 10% and 0.5% serum (FIG. 1C, and data notshown). This was at least partly due to an increase in cells arrested atthe G₁ phase of the cell cycle in 14-3-3 zeta siRNA-treated cells, asshown by fluorescence-activated cell sorter (FACS) analysis of twobreast cancer cell lines, a cervical carcinoma cell line (HeLa), and alung cancer cell line (H1299) after 14-3-3 zeta siRNA treatment (FIG.1D). Further studies were conducted to determine whether 14-3-3 zetasiRNA alters the expression of G₁ phase cell cycle regulators. Comparedto the control siRNA-treated cells, 14-3-3 zeta siRNA-treated cellsexhibited a marked increase in p₂₇ ^(KIP1) and p21^(CIP1) expression,whereas p16^(INK4a) and Cyclin D1 expression were not changedsignificantly except in H1299. Therefore, the G₁ arrest from 14-3-3 zetasilencing may result from up-regulating the cyclin-dependent kinaseinhibitors p27^(KIP1) and p21^(CIP1). Similar results were observed whencells are treated with another 14-3-3 zeta siRNA duplex targeting adifferent mRNA region (data not shown).

Example 6 14-3-3 Zeta is Involved in the Stress-Induced ApoptoticResponse

Studies were conducted to investigate the role of 14-3-3 zeta instress-induced apoptotic responses. Under serum starvation conditions,MCF-7 and HeLa cells treated with 14-3-3 zeta siRNA showed a remarkableincrease in apoptotic annexin-positive (FIG. 2A) and sub-G₁ (FIG. 2B)populations compared to cells treated with control siRNA, indicatingthat silencing of 14-3-3 zeta sensitized these cancer cells tostress-induced apoptosis. To understand the underlying mechanisms, theactivation of two major apoptosis pathways (death receptor pathway andmitochondria pathway; Wajat, 2002) in cancer cells was investigatedafter 14-3-3 zeta siRNA treatment. While no significant difference inFas activation was found between 14-3-3 zeta siRNA and controlsiRNA-treated cells (data not shown), immunostaining of cytochrome Cclearly demonstrated its release from mitochondria in 14-3-3 zetasiRNA-treated MCF-7 cells after serum withdrawal. Immunoblot analysis ofcaspase-9 showed a decrease of pro-caspase-9 in 14-3-3 zetasiRNA-treated cells, indicative of activation of caspase-9 byproteolytic cleavage. An examination of downstream caspase substratessuch as poly (ADP-ribose) polymerase (ARP) and p₁₃₀ ^(CAS) also revealedtheir cleavage in 14-3-3 zeta siRNA-treated cells. Thus, downregulationof 14-3-3 zeta could sensitize cancer cells to apoptosis throughactivation of the mitochondria pathway. Moreover, an increased BADexpression was detected in 14-3-3 zeta siRNA-treated cells. BAD is aknown 14-3-3 ligand and a pro-apoptotic molecule facilitating cytochromeC release. It was therefore proposed that downregulation of 14-3-3 zetaincreases BAD protein levels through yet undefined mechanisms leading toincreased cytochrome C release and subsequent caspase activation.Furthermore, addition of a caspase inhibitor (Z-VAD-FMK) to the 14-3-3zeta siRNA-treated MCF-7 cells in serum-free media effectively blockedPARP cleavage and 14-3-3 zeta siRNA failed to sensitize these cells tostress-induced apoptosis (FIG. 2C). Therefore, the sensitization tostress-induced apoptosis by 14-3-3 zeta siRNA may require sequentialevents, involving the up-regulation of BAD, increased cytochrome Crelease, caspase activation, and caspase substrate cleavages.

Downregulation of 14-3-3 zeta also inhibited the transforming propertiesof cancer cells, as 14-3-3 zeta siRNA-treated MDA-MB-435 and MCF-7 cellsformed less soft agar colonies than the control group (FIG. 2D). Thisindicated the potential of targeting 14-3-3 zeta in blocking malignanttransformation. To test this, MDA-MB-435 cells were transfected with14-3-3 zeta siRNA or control siRNA, and then cells were injected intothe mammary fat pads (mfp) of female SCID mice. The mice in the 14-3-3zeta siRNA group demonstrated markedly delayed tumor onset and reducedtumor growth (FIG. 2E, FIG. 2F), which paralleled with 14-3-3 zetareduction, p27^(KIP1) upregulation, and increased TUNEL positiveapoptotic cells in the tumors grown in the mfp. Thus, a brief blockageof 14-3-3 zeta was sufficient to suppress tumorigenicity of MDA-MB-435breast cancer cell line in vivo.

Example 7 14-3-3 Zeta Expression Provides Prognostic Information inPatients with Cancer

14-3-3 zeta is a biomarker for poor prognosis of breast cancer. Tofurther investigate the impact of 14-3-3 zeta overexpression on cancerprogression in patients, IHC analysis of 14-3-3 zeta was performed inprimary invasive breast carcinomas from 107 patients, who were treatedwith cyclophosphamide, methotrexate, and 5-fluorouracil after mastectomywith a median follow-up time of 62 months (Yang et al., 2002). Amongthese patients' samples, 22 were negative or weakly positive for 14-3-3zeta expression, whereas 89 were moderately or strongly positive, i.e.,had 14-3-3 zeta overexpressions to different extents. Positive 14-3-3zeta signals were observed mainly in the cytoplasm of the breastcarcinoma cells, whereas benign breast epithelial cells and stromalcomponents were negative or weakly positive for 14-3-3 zeta stainingunder the same experimental conditions. Using Kaplan-Meier analyses,high 14-3-3 zeta scores in breast tumors (n=48) are significantlyassociated with reduced disease-free survival (P=0.02) and reducedoverall survival (P=0.01) (FIG. 3). Thus, 14-3-3 zeta is a biomarker forpoor prognosis of breast cancers. Compared to known poor prognosticmarkers such as HER2 or uPA overexpression, 14-3-3 zeta strong positivestaining were observed in more patients (approximately 45% for 14-3-3ζcompared to 30% for HER2 and 15% for uPA (Rogers et al., 2002).Therefore, monitoring 14-3-3 zeta protein levels in breast cancerspecimens may provide additional prognostic information to the currentclinical and pathological parameters.

In this study, it has been demonstrated that 14-3-3 zeta isoverexpressed in multiple human primary tumors and cancer cell linesrepresenting a frequent event in cancer development of a broad spectrumof cancer types. Downregulation of 14-3-3 zeta led to G₁ arrest byupregulation of p27^(KIP1) and p21^(CIP1) in all cancer cells examined,indicating that 14-3-3 zeta may be a key factor utilized by many typesof cancer cells to control certain common pathways during cancerprogression. Mechanisms underlying 14-3-3 zeta ablation-inducedp27^(KIP1) and p21^(CIP1) upregulation are currently underinvestigation. Downregulation of 14-3-3 zeta also sensitizes cancercells to apoptosis, indicating 14-3-3 zeta plays an essential role incancer cell survival and 14-3-3 zeta might be used as a predictivemarker for response to chemotherapy in cancer patients. A brief blockadeof 14-3-3 zeta expression by siRNA (up to 7 days) considerably inhibitedtumor onset and tumor growth from breast cancer cells in vivo,signifying 14-3-3 zeta is an attractive therapeutic target in humancancers. The significant association between 14-3-3 zeta overexpressionand poor survival of breast cancer patients further demonstrates theclinical potential of 14-3-3 zeta as a prognostic marker and atherapeutic target.

Example 8 14-3-3 Zeta Gene Amplification is Correlated with itsOverexpression in Cancer Patients

Based on the study on samples from normal tissues, criteria were set upfor 14-3-3 zeta gene amplification as the following. The upper limitcut-off value was set to the mean+3 SD of the technical variation foundin the analysis of 10 normal breast tissue samples. Samples with lessthan 19% of cells having >2 copies of 14-3-3 zeta gene per cell wereclassified as “normal”; samples with 19% or more cells having >2 copies,the average copy number being between 2 and 5, and the ratio of 14-3-3zeta to chromosome 8 being ≧1 were classified as “low”; samples withmore than 19% cells having >2 copies, the average copy number being ≧5,and the ratio of 14-3-3 zeta to chromosome 8≧1 were regarded as “high.”Forty-three breast cancers were analyzed for the 14-3-3 zeta gene andthe chromosome 8 centromere using dual-color FISH. 11/43 (25.6%) ofsamples were normal, 9/43 (20.9%) of samples were low, and 22/43 (51.2%)of samples showed high amplification. In about 70% of cases, there is acorrelation between 14-3-3 zeta gene amplification and proteinexpression.

Example 9 14-3-3 Zeta Interacts With PI3-Kinase

Methods

14-3-3 zeta stable transfections. To generate 14-3-3 zeta stabletransfectants, one million cells were seeded onto a 100 mm tissueculture plate 24 hours prior to transfection in DMEM/F12 mediumcontaining 10% FBS. MCF-7 and MDA-MB-435 breast cancer and H1299 lungcancer cell lines were used to generate HA-tagged 14-3-3 zeta stabletransfectants. P85 stable transfectants were made by stably transfectingboth HA-14-3-3 zeta and the HIS-tagged p85 contructs in the MCF-7 breastcancer cell line.

10 μg of DNA were added to 750 μl of Opti-MEM I (Invitrogen) containing20 μl of Plus Reagent (Invitrogen), mixed and incubated for 15 minutesat room temperature. At the same time, 30 μl of Lipofectamine(Invitrogen) were mixed and incubated with 750 μl of Opti-MEM I. Afterincubation, the two parts were mixed and incubated for another 15minutes at room temperature. This combination was diluted with 5 ml ofOpti-MEM I and added to cells that were pre-washed once with 5 ml ofOpti-MEM I. Five hours post-transfection, 6.5 ml of DMEM/F12 media with20% FBS were added to the cells. The transfection solution was removed24 hours post-transfection and replaced with DMEM/F12 medium containing10% FBS. At 48 hours post transfection, cells were split to a very lightdensity in 100 mm tissue culture plates and media containing 800 μg/mLof G418 drug selection. Cells were maintained in selection media untilsingle colonies of cells formed in the dish. Multiple colonies wereselected, expanded and screened for the expression of HA-14-3-3 zetaprotein by Western blot analysis. Clones with the highest expressionwere selected and expanded further.

Western Blot Analysis. Proteins were separated and analyzed byharvesting cells in 500 μl or less of 14-3-3 zeta lysis buffer (50 mMTris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 2 mM MgSO₄, 0.1% Tween-20,protease cocktail inhibitor, 1 mM PMSF, 20 mM NaF, 1 mM Na₃VO₄, 20 mMβ-glycerophosphate) and allowed to incubate on ice for 20 minutes orstored at −80° C. until used. Cells were further lysed by six passesthrough a 22 gauge needle. Cellular debris was pelleted bycentrifugation at 14000 rpm for 15 minutes at 4° C. The supernatant wastransferred to a new tube and the Bradford Method determined proteinconcentration. The indicated amount of protein lysate was resuspended in6×SDS lysis buffer and heated at 100° C. for 5 minutes. The lysate wasthen separated by SDS-PAGE. The proteins were transferred to anitrocellulose membrane and probed with the respective antibodies.

Immunoprecipitation. To determine protein—protein association, cellswere harvested in 14-3-3 zeta lysis buffer (50 OM Tris-HCl pH 7.5, 150mM NaCl, 2 mM EGTA, 2 mM MgSO₄, 0.1% Tween-20, protease cocktailinhibitor, 1 mM PMSF, 20 mM NaF, 1 mM Na₃VO₄, 20 mM β-glycerophosphate)and allowed to incubate on ice for 20 minutes. Cells were further lysedby six passes through a 22 gauge needle. Cellular debris was pelleted bycentrifugation at 14000 rpm for 15 minutes at 4° C. The supernatant wastransferred to a new tube and the Bradford Method determined proteinconcentration. Immunoprecipitation (IP) was performed by adding theindicated antibodies to 1 mg of total cellular lysate which wasincubated overnight at 4° C. In some cases, cell lysates werepre-cleared by incubating 30 μl of protein G beads with lysate for 1hour and recovering lysate to a new tube. After incubation withantibody, 40 μl of protein G agarose beads were added and incubated for2 hours more to pull down immune complexes. Beads with attachedantibodies and proteins were recovered by centrifugation at 5000 rpm at4° C. followed by five washes with lysis buffer. The bead complexes werethen used in other assays or resuspended in 30 μl of lysis buffer with 5μl of 6×SDS loading dye and analyzed by Western blot analysis.

PI3K Assay. To determine the activity of PI3K from MCF-7 cells treatedwith siRNA, cells were serum starved for 18 hours. Where indicated,cells were re-stimulated with 20 ng/ml of Heregulin for 10 minutes.Cells were lysed and immunoprecipitated with 1 μg of phosphotyrosine-20antibody (Signal Transduction Laboratories, Inc.). Immune complexes werewashed three times with lysis buffer and once with kinase buffer (20 mMTris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA). The beads wereresuspended in 50 μl of kinase buffer with 0.2 mg/ml ofphosphatidylinositol. Then 20 μCi of [γ-³²P]ATP and 20 mM MgCl₂ wereadded for 10 minutes at room temperature. Reactions were terminated byadding 150 μl of chloroform/methanol, and phosphatidylinositol wasextracted with 100 μl of chloroform. The organic phase was washed withmethanol/1M HCL (1:1) and lyophilized. Following reuspension in 15 μl ofchloroform, phosphatidylinositol was spotted on a silica gel 60 thinlayer chromatography plate and resolved in chloroform/methanol/28%ammonium hydroxide/water (86:76:10:14) for 45 minutes. Phosphorylatedproducts were visualized by autoradiography.

Induction of Akt activity. To determine the contribution of 14-3-3 zetato Akt activation, 1×10⁶ cells stably expressing full length 14-3-3 zetaor C-terminal deleted 14-3-3 zeta were plated in 100 mm tissue cultureplates and allowed to attach overnight. Cells were washed three timeswith serum free media and replenished with serum free media. Cells werestarved for 18 hours and either left unstimulated or stimulated for 10minutes with media containing 10% FBS. Treated cells were washed oncewith 5 mL of 1×PBS and harvested in 14-3-3 zeta lysis buffer. Lysateswere analyzed as in Western blot analysis with the indicatedphospho-specific antibodies and reprobed with the native antibody as aloading control.

Site Directed Mutagenesis of p85. To change the amino acid serine 83 toalanine on p85, a p85 expression vector containing the p85 cDNA was usedas the mutagenesis template. The QuickChange site directed mutagenesiskit from Stratagene (La Jolla, Calif.) was used to carry out themutagenesis of p85 according to the manufactures protocol. Sequencing ofthe mutated plasmid was used to confirm the amino acid change in theexpression plasmid.

TUNEL Assay for measurement of apoptosis. For analysis of apoptosisusing terminal deoxynucleotide transferase dUTP nick end labeling(TUNEL), 6×10⁵ cells were plated in 60 mm tissue culture plates andallowed to attach overnight. Apoptosis was induced in the cells by serumfree conditions and collected at the designated times. Cells wereharvested into a 15 mL tube by collecting the media from the plate,which includes floating cells, and trypsinizing the attached cells.Cells were pelleted by centrifugation at 1200 rpm. The pellet was washedwith 3 mL of 1×PBS and then resuspended in 1% paraformaldehyde in PBSand placed on ice for 30 minutes. Cells were pelleted and washed with1×PBS and then resuspended in 70% ethanol and placed overnight at −20°C. Apoptosis was detected using the APO-BRDU kit from Phoenix FlowSystems (San Diego, Calif.) following the manufacturer's protocol.Briefly, fixed cells were pelleted and washed twice with 1 mL of Washbuffer. Cells were then resupended in 50 μl of DNA Labeling Solution(TdT reaction buffer, Tdt enzyme and Br-dUTP) and incubated at 37° C.for 3 hours. After incubation, 1 mL of Rinse buffer was added to thereaction and cells were pelleted and washed again in Rinse buffer. Thecells were resupended in 100 μl of Antibody Solution and incubated inthe dark at room temperature for 2 hours. Cells were stained withPropidium Iodide/RNase A solution and analyzed by flow cytometry.

Results

14-3-3 zeta associates with phosphatidylinositol-3-kinase (PI3K) incancer cells. The previous results indicate that blocking 14-3-3 zeta incancer cells by siRNA sensitizes these cells to apoptosis and reducestheir growth. This indicates that 14-3-3 zeta mediates an essentialsurvival and growth signal within cancer cells.Phosphatidylinositol-3-kinase (PI3K) and its down stream signalingmolecule Akt regulate apoptosis and cell growth in many cell types(Vivanco and Sawyers, 2002). Other studies using hematopoietic cellshave suggested 14-3-3 may modulate PI-3K; however, the role of 14-3-3 inregulating PI3K is not established in cancer cells (Guthridge et al.,2000; Munday et al., 2000). Therefore, it is hypothesized that 14-3-3zeta could associate with PI3K and modulate PI3K activity in humancancer cells to mediate survival signals and growth.

Immunoprecipitation of HA-14-3-3 zeta from MCF-7 cells stablytransfected with a HA-14-3-3 zeta expression vector pulled down theendogenous p85 subunit in 10% serum conditions but not in cells deprivedof serum (FIG. 4A, left). It was then determined if this association wasserum dependent by starving these cells and then re-stimulating themwith 10% serum. Association of HA-14-3-3 zeta and endogenous p85 couldbe restored 10 minutes after serum addition (FIG. 4A, left). This samepattern was also demonstrated in the H1299 lung cancer cell line stablytransfected with HA-14-3-3 zeta (FIG. 4A, right). In addition,immunoprecipitation of either the endogenous p85 or p110 subunitdemonstrated association with HA-14-3-3 zeta (FIG. 4B). The resultsindicate that 14-3-3 zeta associates with PI-3K in a serum dependentmanner and suggests 14-3-3 zeta may be involved in PI3K activation bygrowth factors in the serum.

14-3-3 zeta modulated phosphatidylinositol-3-kinase (PI3K) activity. Theassociation of p85 regulatory subunit and p110 catalytic subunit isrequired for PI3K activity (Cantley, 2002) and 14-3-3 binding to PI3Kmay modulate its activity. Therefore, it was hypothesized that 14-3-3zeta association with p85 could enhance PI3K kinase activity in breastcancer cells. To investigate if PI-3K activity was modulated by 14-3-3zeta, MCF-7 cells were transfected with control or 14-3-3 zeta siRNA.Cells were starved and then stimulated with the growth factor,Heregulin. MCF-7 14-3-3 zeta siRNA treated cells indeed exhibitedreduced activation of PI3K in response to Heregulin stimulation comparedto control siRNA treated cells (FIG. 4C). The data indicated thatsilencing of 14-3-3 zeta reduced PI-3K activation by growth factor.

Blocking 14-3-3 zeta inhibited Akt activation. PI3K is a well-knownupstream activator of the survival signaling kinase, Akt (Vivanco andSawyers, 2002). The Akt kinase is known to regulate signaling pathwaysinvolved in cell survival and proliferation (Vivanco and Sawyers, 2002).PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) tophosphatidylinositol 3,4,5-trisphosphate (PIP3) in the cell membrane.The lipid by-product PIP3, generated by PI3K, activates Akt (Cantley,2002). These results demonstrate that blocking 14-3-3 zeta reduces PI3Kactivity thereby reducing the production of PIP3 in the cells.Therefore, downregulation of 14-3-3 zeta may affect downstream cellsurvival pathways by inhibiting Akt activity. To test this hypothesis,MDA-MB-435 and MCF-7 cells were transfected with control or 14-3-3 zetasiRNA, serum starved, or starved then stimulated with 10% serun forvarious times (FIG. 4D). Activation of Akt, as measured by Aktphosphorylation using phospho-serine473-Akt specific antibody, wasreduced by 14-3-3 zeta siRNA compared to the control MDA-MB-435 cells.In the controls, Akt was dramatically activated 20 minutes and remainedelevated after serum addition compared to the basal (+). In contrast,activation of Akt in cells treated with 14-3-3 zeta siRNA was minimal at20 minutes and did not increase by 45 minutes. A similar trend wasdemonstrated in the MCF-7 cell line. In the MCF-7 controls, Akt wasactivated 20 minutes after serum addition and remained elevated up to 60minutes. In contrast, activation of Akt was reduced during this sametime period in 14-3-3 zeta siRNA treated cells. These results indicatethat 14-3-3 zeta regulates the signaling pathway involved in Aktactivation partly by regulating the kinase activity of PI3K.

Serine 83 of p85 is necessary for 14-3-3 zeta association, PI3Kactivation and anti-apoptosis. It was next determined whether 14-3-3zeta binding to p85 is critical for 14-3-3 zeta's effects on PI3K/Aktactivation and anti-apoptosis by disrupting the binding of 14-3-3 zetato p85. Most 14-3-3 zeta binding partners contain at least one consensus14-3-3 zeta binding site that consists of a phosphorylated serineresidue (Rittinger et al., 1999). Analysis of the p85 protein sequencerevealed a possible consensus 14-3-3 zeta binding site surroundingserine 83 of p85. Using site directed mutagenesis, serine 83 of p85 wasmutated to alanine (S83A). MCF-7 cells expressing HA-14-3-3 were thenstably transfected with expression vectors encoding histidine taggedwild type p85 (p85^(wt)) and p85 with serine 83 to alanine (p85^(S83A)).Expression of these constructs in MCF-7 cells achieved proteins levelssimilar to endogenous p85 (FIG. 5A). To determine if association wasreduced between 14-3-3 zeta and p₈₅ ^(S83A), stable MCF-7 cells wereserum starved for 18 hours and then stimulated with 10% serum media for10 minutes. Cells were lysed and immunoprecipated (IP) with antibodiesto p85 and to HIS-tag on p85, then western blot with anti-HIS andanti-HA to determine IP efficiency and 14-3-3 zeta binding. HA-14-3-3zeta association with p85^(S83A) was greatly reduced compared top85^(WT) in both p85 and HIS IP (FIG. 5B). The data indicate that 14-3-3zeta associates with p85, at least partly through binding to serine 83on p85. Furthermore, mutation of serine 83 on p85 reduced activation ofPI3K by 35% compared to p₈₅ ^(WT) in cells maintained in 10% serum media(FIG. 5C). In addition, activation of Akt was reduced in p₈₅ ^(S83A)cells maintained in 10% serum media (FIG. 5D). Apoptosis induced byserum starvation was also elevated in cells expressing p85^(S83A)compared to p85^(WT) (FIG. 5E). These results indicate that 14-3-3 zetaassociates with p85 through serine 83 thereby modulating PI3K/Aktactivation and cell survival in response to stress induced apoptosis.

14-3-3 zeta interacts with both the p85 and p110 subunits of PI3-kinase.Interaction between 14-3-3 zeta and PI3-kinase were examined byimmunoprecipitation followed by western blot analysis. MCF-7 and H1299cells were stably transfected with an HA-14-3-3 zeta expression vector.Immunoprecipitation of HA-tagged 14-3-3 zeta effectively brought downp85 subunit in a phosphorylation-dependent manner. On the other hand,immunoprecipitation of either p85 or p110 subunit also brought downHA-14-3-3 zeta, as revealed by western blot using HA antibodies.

Example 10 Assays to Identify Modulators of Death of a Cancer Cell

Using the teachings of the specification and the knowledge of thoseskilled in the art, one can conduct assays to identify modulators ofdeath of a cancer cell. Candidate substances suspected of modulating14-3-3 zeta activity can first be identified. The candidate substancemay include, but are not limited to, small molecules, peptides,polypeptides, proteins, nucleic acids, or any molecule suspected ofmodulating 14-3-3 zeta activity. The candidate substance may then becontacted with the cancer cell, and modulation of death of the cancercell can be measured. Any method known to those of skill in the art canbe used to contact the candidate substance with the cancer cell. Forexample, the candidate substances can be applied to a culture of cancercells in vitro. Alternatively, animal models of cancer well known tothose of skill in the art can be used in these assays to identifymodulators of death of a cancer cell. Modulation of cell death caninclude either an increase in cell death, or an inhibition of cell deathrelative to a control cell that has not been contacted with thecandidate substance.

Example 11 Clinical Trials of the Use of Modulators of 14-3-3 ZetaExpression and/or Function in the Treatment of Diseases in General

This example is generally concerned with the development of humantreatment protocols using modulators of 14-3-3 zeta expression and/orfunction in the treatment of diseases, such as hyperproliferativediseases. Examples of these diseases include cancer, such as breastcancer. A more detailed example pertaining to cancer is discussed in thenext example.

The various elements of conducting a clinical trial, including patienttreatment and monitoring, will be known to those of skill in the art inlight of the present disclosure. The following information can be usedas a general guideline for use in administering modulators of 14-3-3zeta expression and/or function in clinical trials.

Patients with the targeted disease can be newly diagnosed patients orpatients with existing disease. Patients with existing disease mayinclude those who have failed to respond to at least one course ofconventional therapy.

The modulator of 14-3-3 zeta activity may be a substance that eitherinhibits 14-3-3 zeta expression and/or function, or alternatively may bea substance that promotes 14-3-3 zeta expression and/or function. Themodulator may be administered alone or in combination with anothertherapeutic agent. The agents may be administered intravenously, orally,topically, intratumorally, or by another mechanism that is specific tothe disease that is being treated. The agent may be administered duringa procedure, such as intraoperatively.

One of ordinary skill in the art would determine an appropriate startingdose, or the starting dose would be dictated by a clinical protocol.Dose escalation may be done by 100% increments until drug relatedtoxicity of a specific level develops. Thereafter dose escalation mayproceed by 25% increments. The administered dose may be fractionated.

The modulator of 14-3-3 zeta activity may be administered over a shortinfusion time or at a steady rate of infusion over a period of days. Themodulator may be administered alone or in combination with other agents.The infusion given at any dose level will be dependent upon the toxicityachieved after each.

Physical examination, laboratory tests, and other clinical studiesspecific to the disease being treated may, of course, be performedbefore treatment and at intervals of about 3-4 weeks later. Laboratorystudies can include CBC, differential and platelet count, urinalysis,SMA-12-100 (liver and renal function tests), coagulation profile, andany other appropriate chemistry studies to determine the extent ofdisease, or determine the cause of existing symptoms. If necessary,appropriate biological markers in serum can be monitored.

Example 12 Clinical Trials of the Use of Modulators of 14-3-3 ZetaActivity in the Treatment of Cancer

This example is concerned with the development of human treatmentprotocols using modulators of 14-3-3 zeta activity in the treatment ofcancer. The various elements of conducting a clinical trial, includingpatient treatment and monitoring, will be known to those of skill in theart in light of the present disclosure. The following information can beused as a general guideline for use in administering modulators of14-3-3 zeta activity in clinical trials pertaining to cancer treatment.

Patients with cancer chosen for clinical study will typically havefailed to respond to at least one course of conventional therapy.Measurable disease is not required.

The modulator of 14-3-3 zeta activity may be administered alone or incombination with another chemotherapeutic agent. The administration maybe intravenously, directly into the tumor, topically, or in any othermanner known to those of skill in the art. The starting dose may be 0.5mg/kg body weight. Three patients may be treated at each dose level inthe absence of grade >3 toxicity. Dose escalation may be done by 100%increments (0.5 mg, 1 mg, 2 mg, 4 mg) until toxicity is detected.Thereafter dose escalation may proceed by 25% increments.

The modulator of 14-3-3 zeta activity may be administered over a shortinfusion time or at a steady rate of infusion over a 7 to 21 day period.The modulator may be administered alone or in combination with theanti-cancer drug. The infusion given at any dose level will be dependentupon the toxicity achieved after each. Increasing doses of the modulatorof 14-3-3 zeta activity in combination with an anti-cancer drug will beadministered to groups of patients until approximately 60% of patientsshow unacceptable toxicity. Doses that are 2/3 of this value could bedefined as the safe dose.

Physical examination, tumor measurements, and laboratory tests can, ofcourse, be performed before treatment and at intervals of about 3-4weeks later. Laboratory studies should include CBC, differential andplatelet count, urinalysis, SMA-12-100 (liver and renal function tests),coagulation profile, and any other appropriate chemistry studies todetermine the extent of disease, or determine the cause of existingsymptoms. Also appropriate biological markers in serum can be monitored.

To monitor disease course and evaluate the anti-tumor responses, it iscontemplated that the patients may be examined for appropriate tumormarkers every 4 weeks, if initially abnormal. Laboratory studies such asa CBC, differential and platelet count, coagulation profile, and/orSMA-12-100 shall be performed weekly. Appropriate clinical studies suchas radiological studies should be performed and repeated every 8 weeksto evaluate tumor response.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabledisease for at least a month. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules or at least 1month with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater with progressionin one or more sites.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. 4,578,770-   U.S. Pat. 4,596,792-   U.S. Pat. 4,599,230-   U.S. Pat. 4,599,231-   U.S. Pat. 4,601,903-   U.S. Pat. 4,608,251-   U.S. Pat. 4,797,368-   U.S. Pat. 5,139,941-   U.S. Pat. 5,578,832-   U.S. Pat. 5,861,242-   U.S. Pat. 5,994,131-   U.S. Pat. 6,406,921-   U.S. Pat. 6,457,809-   Abbondanzo, Ann. Diagn. Pathol., 3(5):318-327, 1999.-   Aitken et al., Biochem. Soc. Trans., 30(4):351-360, 2002.-   Aitken et al., Nature, 344(6267):594, 1990.-   Aitken et al., Trends Biochem. Sci., 17:498-501, 1992.-   Aitken, Trends Cell Biol., 6(9):341-347, 1996.-   Albertson, Breast Cancer Res. Treat., 78(3):289-298, 2003.-   Allred et al., Arch Surg, 125(1):107-13, 1990.-   Bernard and Wittwer, Clinical Chemistry, 48:1178-1185, 2002.-   Boussif et al., Proc. Natl. Acad. Sci. USA, 92(16):7297-7301, 1995.-   Brown et al. Immunol. Ser., 53:69-82, 1990.-   Brunet et al., Cell, 96(6):857-868, 1999.-   Caley et al., J. Virology, 71(4):3031-3038, 1997.-   Cantley, Science, 296:1655-1657, 2002.-   Chan et al., Biochem. Biophys. Res. Commun., 270(2):581-587, 2000.-   Chang et al., Hepatology, 14:134A, 1991.-   Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.-   Coffin, In: Virology, Fields et al. (Eds.), Raven Press, N.Y.,    1437-1500, 1990.-   Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.-   Davis et al, Curr. Biol., 6:146-148, 1996.-   De Valck et al., Biochem. Biophys. Res. Commun., 238(2):590-594,    1997.-   Elbashir et al., Methods, 26:199-213, 2002.-   Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.-   Ferguson et al., Proc. Natl. Acad. Sci. USA, 97(11):6049-6054, 2000.-   Fodor et al., Biochemistry, 30(33):8102-8108, 1991.-   Forbes, Semin. Oncol., 24(1 Suppl 1):S1-5-S1-19; S20-S35, 1997.-   Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.-   Fry et al., Science, 265(5175):1093-1095, 1994.-   Gopal, Mol. Cell. Biol., 5:1188-1190, 1985.-   Graham and Prevec, Biotechnology, 20:363-390, 1992.-   Graham and Van Der Eb, Virology, 52:456-467, 1973.-   Graham et al, J. General Virology, 36:59-74, 1977.-   Grunhaus et al., Seminar in Virology, 200(2):535-546, 1992.-   Guthridge et al., Mol. Cell, 6(1):99-108, 2000.-   Hacia et al., Nature Genet., 14:441-449, 1996.-   Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985.-   Hermeking et al., Mol. Cell, 1(1):3-11, 1997.-   Horwich et al., J. Virology, 64:642-650, 1990.-   Ichimura et al., J. Neurochem., 56(4):1449-1451, 1991.-   Ichimura et al., Proc. Natl. Acad. Sci. USA, 85(19):7084-7088, 1988.-   Isobe et al., J. Mol. Biol., 217(1):125-132, 1991.-   Jones and Shenk, Cell, 13:181-188, 1978.-   Kallionieemi et al., Science, 258(5083):818-821, 1992.-   Kerr et al., Br. J. Cancer, 26(4):239-257, 1972.-   Konigshoff et al., Clinical Chemistry, 49:219-229, 2003.-   Laughlin et al., J. Virol., 60(2):515-524, 1986.-   Lebkowski et al., Mol. Cell. Biol., 8(10):3988-3996, 1988.-   Liu et al., J. Biol. Chem., 271(24):14591-14595, 1996.-   Liu et al., Nature, 376(6536):191-194, 1995.-   MacBeath and Schreiber, Science, 289:1760-1763, 2000.-   Masters and Fu, J. Biol. Chem., 276(48):45193-45200, 2001.-   McLaughlin et al., J. Virol., 62(6):1963-1973, 1988.-   Moore and Perez, In: Physiological and biochemical aspects of    nervous integration, Carlson (Ed.,) 343-359, Prentice-Hall, N.J.,    1967.-   Munday et al., Blood, 96(2):577-584, 2000.-   Muzyczka, Curr. Topics Microbiol. Immunol., 158:97-129, 1992.-   Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.-   Pandey and Mann, Nature, 405:837-846, 2000.-   PCT Appln. WO 84/03564-   Pease et al., Proc. Natl. Acad. Sci. USA, 91:5022-5026, 1994.-   Pelletier and Sonenberg, Nature, 334:320-325, 1988.-   Pinkel et al., Nat. Genet., 20(2):207-211,.1998.-   Pollack et al., Nat. Genet., 23(1):41-46, 1999.-   Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.-   Remington's Pharmaceutical Sciences, 15^(th) ed., pages 1035-1038    and 1570-1580, Mack Publishing Company, Easton, Pa., 1980.-   Renan, Radiother. Oncol., 19:197-218, 1990.-   Ries et al., Clin. Cancer Res., 5(5):1115-1124, 1999.-   Rippe et al., Mol. Cell Biol., 10:689-695, 1990.-   Rittinger et al., Mol. Cell, 4:153-166, 1999.-   Rogers et al., Eur. J. Surg. Oncol., 28(5):467-478, 2002.-   Rosenquist et al., J. Mol. Evol., 51(5):446-458, 2000.-   Roux et al., Proc. Natl. Acad. Sci. USA, 86:9079-9083, 1989.-   Shin et al., Biochem. Biophys. Res. Commun., 246(2):313-319, 1998.-   Shoemaker et al., Nature Genet., 14:450-456, 1996.-   Snijders et al., Mol. Pathol., 53(6):289-294, 2000.-   Solinas-Toldo et al., Genes Chromosomes Cancer, 20(4):399-407, 1997.-   Sondik, Cancer, 74(3 Suppl):995-999, 1994.-   Tan et al., Mol. Cell, 9:993-1004, 2002.-   Toker et al., Eur. J. Biochem., 191(2):421-429, 1990.-   Top et al., J. Infect. Dis., 124:155-160, 1971.-   Tratschin et al., Mol. Cell. Biol., 4:2072-2081, 1984.-   Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986.-   Veltman et al., Cancer Res., 63(11):2872-2880, 2003.-   Vercoutter-Edouart et al., Cancer Res., 61(1):76-80, 2001.-   Vivanco and Sawyers, Nat. Rev. Cancer, 2:489-501, 2002.-   Wajant, Science, 296:1635-1636, 2002.-   Walker, Science, 296:557-559, 2002.-   Wang and Shakes, J. Mol. Evol., 43(4):384-398, 1996.-   Wang et al., Anal. Chem., 72(21):5285-5289, 2000.-   Watanabe et al., Brain Res. Mol. Brain Res., 10(2):151-158, 1991.-   Welch and Wei, Endocrine-Related Cancer, 5:155-197, 1998.-   Wu and Wu, Biochemistry, 27:887-892, 1988.-   Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.-   Xiao et al., Nature, 376(6536):188-191, 1995.-   Yaffe, FEBS Lett., 513(1):53-57, 2002.-   Yang et al. Cancer Res., 61:8150-8157, 2001.-   Yang et al., Molec. Pharmacol., 61:269-276, 2002.-   Yang et al., Proc Natl. Acad. Sci. USA, 87:9568-9572, 1990.-   Zha et al., Cell, 87(4):619-628, 1996.-   Zhang et al., Proc. Natl. Acad. Sci. USA, 96(15):8511-8515, 1999.

1. A method of determining prognosis in a subject with breast cancer,comprising determining overexpression of 14-3-3 zeta protein (SEQ IDNO:1) in a breast tumor sample from said subject relative to expressionof 14-3-3 zeta protein in breast tissue from a subject that does nothave breast cancer, wherein said overexpression of 14-3-3 zeta proteinindicates that said subject with breast cancer has a lower survival rateas compared to a subject with a breast cancer that does not overexpress14-3-3 zeta protein.
 2. The method of claim 1, wherein the subject withbreast cancer is a human.
 3. The method of claim 1, wherein determiningexpression of 14-3-3 zeta protein is measured by western blot analysis,immunohistochemistry, and/or protein array.
 4. The method of claim 1,wherein the breast cancer is metastatic breast cancer.
 5. The method ofclaim 3, wherein determining expression of 14-3-3 zeta protein ismeasured by western blot analysis.
 6. The method of claim 3, whereindetermining expression of 14-3-3 zeta protein is measured byimmunohistochemistry.
 7. The method of claim 3, wherein determiningexpression of 14-3-3 zeta protein is measured by protein array.
 8. Amethod of diagnosing breast cancer, lung cancer, liver cancer, uterinecancer, or stomach cancer in a subject, comprising determiningoverexpression of 14-3-3 zeta protein (SEQ ID NO:1) in a breast, lung,liver, uterine, or stomach tissue test sample, respectively, from saidsubject relative to expression of 14-3-3 zeta protein in breast, lung,liver, uterine, or stomach tissue control sample from a subject thatdoes not have breast cancer, lung cancer, liver cancer, uterine cancer,or stomach cancer, respectively, wherein said overexpression of 14-3-3zeta protein indicates that the patient from which the test sample wasobtained has breast cancer, lung cancer, liver cancer, uterine cancer,or stomach cancer, respectively.
 9. The method of claim 8, wherein thesubject is a human.
 10. The method of claim 8, wherein determiningoverexpression of 14-3-3 zeta protein is measured by western blotanalysis, immunohistochemistry, and/or protein array.
 11. The method ofclaim 10, wherein determining expression of 14-3-3 zeta protein ismeasured by western blot analysis.
 12. The method of claim 10, whereindetermining expression of 14-3-3 zeta protein is measured byimmunohistochemistry.
 13. The method of claim 10, wherein determiningexpression of 14-3-3 zeta protein is measured by protein array.
 14. Themethod of claim 8, wherein the method is a method of diagnosing breastcancer, wherein the tissue test sample is breast tissue.
 15. The methodof claim 8, wherein the method is a method of diagnosing lung cancer,wherein the tissue test sample is lung tissue.
 16. The method of claim8, wherein the method is a method of diagnosing liver cancer, whereinthe tissue test sample is liver tissue.
 17. The method of claim 8,wherein the method is a method of diagnosing uterine cancer, wherein thetissue test sample is uterine tissue.
 18. The method of claim 8, whereinthe method is a method of diagnosing stomach cancer, wherein the tissuetest sample is stomach tissue.